Inhibitors of cruzipain and other cysteine proteases

ABSTRACT

Compounds of general formula (I): wherein R 1 , R 2 , Y, (X) o , (W) n , (V) m , Z and U are as defined in the specification, are inhibitors of cruzipain and other cysteine protease inhibitors and are useful as therapeutic agents, for example in Chagas&#39; disease, or for validating therapeutic target compounds.

[0001] THIS INVENTION relates to compounds which are inhibitors of theprotease cruzipain, a gene product of the Trypanosoma cruzi parasite. Inparticular, the invention provides compounds that are useful for thetherapeutic treatment of Trypanosoma cruzi infection, to the use ofthese compounds, and to pharmaceutical compositions comprising them.Furthermore, this invention relates to compounds which are inhibitorsacross a broad range of cysteine proteases, to the use of thesecompounds, and to pharmaceutical compositions comprising them. Suchcompounds are useful for the therapeutic treatment of diseases in whichparticipation of a cysteine protease is implicated.

[0002] The trypanosomal family of parasites have a substantial worldwideimpact on human and animal healthcare (McKerrow, J. H., et al, Ann. Rev.Microbiol. 47, 821-853, 1993). One parasite of this family, Trypanosomacruzi, is the causative agent of Chagas' disease, which affects inexcess of twenty million people annually in Latin and South America, isthe leading cause of heart disease in these regions and results in morethan 45,000 deaths per annum (Centers for Disease Control and preventionwebsite). In addition, with the increase in migration of the infectedpopulation from rural to urban sites and movements from South andCentral America into North America, the infection is spreading via bloodtransfusions, and at birth. The present treatments of choice forTrypanosoma cruzi infection, nifurtimox and benznidazole (an NADHfumarate reductase inhibitor, Turrens, J F, et al, Mol BiochemParasitol., 82(1), 125-9, 1996) are at best moderately successful,achieving ˜60% cure during the acute phase of infection (see Docampo, R.Curr. Pharm. Design, 7, 1157-1164, 2001 for a general discussion) whilstnot being prescribed at all during the chronic phase wherecardiomyopathy associated heart failure often occurs (Kirchhoff, L. V.New Engl. J. Med., 329, 639-644, 1993). Additionally, these two drugshave serious adverse toxic effects, requiring close medical supervisionduring treatment, and have been shown to induce chromosomal damage inchagastic infants (Gorla, N. B. et al, Mutat. Res. 206, 217-220, 1988).Therefore, a strong medical need exists for new effective drugs for thechemotherapeutic treatment of Trypanosoma cruzi infection.

[0003] Classically, the identification of enzymes found to be crucialfor the establishment or propagation of an infectious disease has beeninstrumental in the development of successful drugs such as antivirals(e.g. HIV aspartyl protease inhibitors) and anti-bacterials (e.g.β-lactam antibiotics). The search for a similar Achilles heel inparasitic infections has examined numerous enzymes (e.g. parasiticdihydrofolate reductase, see Chowdhury, S. F. et al, J. Med. Chem.,42(21), 4300-4312, 1999; trypanothione reductase, see Li, Z. et al,Bioorg. Med. Chem. Lett., 11(2), 251-254, 2001; parasiticglyceraldehydes-3-phosphate dehydrogenase, see Aranov, A. M. et al, J.Med. Chem., 41(24), 4790-4799, 1998). A particularly promising area ofresearch has identified the role of cysteine proteases, encoded by theparasite, that play a pivotal role during the life cycle of the parasite(McKerrow, J. H., et al, Bioorg. Med. Chem., 7, 639-644, 1999).Proteases form a substantial group of biological molecules which to dateconstitute approximately 2% of all the gene products identifiedfollowing analysis of several genome sequencing programmes (e.g. seeSouthan, C. J. Pept. Sci, 6, 453-458, 2000). Proteases have evolved toparticipate in an enormous range of biological processes, mediatingtheir effect by cleavage of peptide amide bonds within the myriad ofproteins found in nature. This hydrolytic action is performed byinitially recognising, then binding to, particular three-dimensionalelectronic surfaces displayed by a protein, which aligns the bond forcleavage precisely within the protease catalytic site. Catalytichydrolysis then commences through nucleophilic attack of the amide bondto be cleaved either via an amino acid side-chain of the proteaseitself, or through the action of a water molecule that is bound to andactivated by the protease. Proteases in which the attacking nucleophileis the thiol side-chain of a Cys residue are known as cysteineproteases. The general classification of ‘cysteine protease’ containsmany members found across a wide range of organisms from viruses,bacteria, protozoa, plants and fungi to mammals.

[0004] Biological investigation of Trypanosoma cruzi infection hashighlighted a number of specific enzymes that are crucial for theprogression of the parasite's life cycle. One such enzyme, cruzipain, acathepsin L-like cysteine protease, is a clear therapeutic target forthe treatment of Chagas' disease ((a) Cazzulo, J. J. et al, Curr. Pharm.Des. 7, 1143-1156, 2001; (b) Caffrey, C. R. et al, Curr. Drug Targets,1, 155-162, 2000). Although the precise biological role of cruzipainwithin the parasite's life cycle remains unclear, elevated cruzipainmessenger RNA levels in the epimastigote developmental stage indicate arole in the nutritional degradation of host-molecules in lysosomal-likevesicles (Engel, J. C. et al, J. Cell. Sci, 1597-606, 1998). Thevalidation of cruzipain as a viable therapeutic target has been achievedwith increasing levels of complexity. Addition of a general cysteineprotease inhibitor, Z-Phe-Ala-FMK to Trypanosoma cruzi-infectedmammalian cell cultures blocked replication and differentiation of theparasite, thus arresting the parasite life cycle (Harth, G., et al, Mol.Biochem. Parasitol. 58, 17-24, 1993). Administration of a vinylsulphone-based inhibitor in a Trypanosoma cruzi-infected murine animalmodel not only rescued the mice from lethal infections, but alsoproduced a complete recovery (Engel, J. C. et al, J. Exp. Med, 188(4),725-734, 1998). Numerous other in vivo studies have confirmed thatinfections with alternative parasites such as Leishmania major (Selzer,P. M. et al, Proc. Nat'l. Acad. Sci. U.S.A., 96, 11015-11022, 1999),Schistosoma mansoni and Plasmodium falciparium (Olson, J. E. et al,Bioorg. Med. Chem., 7, 633-638, 1999) can be halted or cured bytreatment with cysteine protease inhibitors.

[0005] A variety of synthetic approaches have been described towards thedesign of cruzipain inhibitors. However, although providing a biological‘proof-of-principle’ for the treatment of Trypanosoma cruzi infection,current inhibitors exhibit a number of biochemical and physicalproperties that may preclude their clinical development. (e.g. see (a)Brinen, L. S. et al, Structure, 8, 831-840, 2000, peptidomimetic vinylsulphones, possible adverse mammalian cell toxicity (see McKerrow, J. H.and Engel, J. unpublished results cited in Scheidt, K. A. et al, Bioorg.Med. Chem, 6, 2477-2494, 1998); (b) Du, X. et al, Chem. Biol., 7,733-742, 2000, aryl ureas, generally with low μM activity, and highClogP values, thus poor aqueous solubility and probably low oralbioavailability; (c) Roush, W. R. et al, Tetrahedron, 56, 9747-9762,2000, peptidyl epoxysuccinates, irreversible inhibitors, with potentactivity verses house-keeping mammalian proteases such as cathepsin B;(d) Li, R. et al, Bioorg. Med. Chem. 4(9), 1421-1427, 1996,bisarylacylhydrazides and chalcones, polyhydroxylated aromatics; (e)U.S. Pat. No. 6,143,931, WO 9846559, non-peptide α-ketoamides). Of themany different approaches to enzyme inhibition to date, only thecruzipain protease inhibitors have proven effective in curingdisease-related animal models of Trypanosoma cruzi infection. Therefore,a clear medical need exists to progress these ‘proof-of-principle’findings towards clinical candidates, suitable for human use, throughthe discovery of more efficacious cruzipain inhibitors that have adesirable combination of potency, selectivity, low toxicity andoptimised pharmacokinetic parameters.

[0006] Cruzipain and indeed many other crucial parasitic proteasesbelong to the papain-like CA C1 family and have close structuralmammalian homologues. Cysteine proteases are classified into ‘clans’based upon a similarity in the three-dimensional structure or aconserved arrangement of catalytic residues within the protease primarysequence. Additionally, ‘clans’ are further classified into ‘families’in which each protease shares a statistically significant relationshipwith other members when comparing the portions of amino acid sequencewhich constitute the parts responsible for the protease activity (seeBarrett, A. J. et al, in ‘Handbook of Proteolytic Enzymes’, Eds.Barrett, A. J., Rawlings, N. D., and Woessner, J. F. Publ. AcademicPress, 1998, for a thorough discussion). To date, cysteine proteaseshave been classified into five clans, CA, CB, CC, CD and CE (Barrett, A.J. et al 1998). A protease from the tropical papaya fruit ‘papain’ formsthe foundation of clan CA, which currently contains over 80 distinct andcomplete entries in various sequence databases, with many more expectedfrom the current genome sequencing efforts. Proteases of clan CA/familyCl have been implicated in a multitude of disease processes e.g. humanproteases such as cathepsin K (osteoporosis), cathepsin S (autoimmunedisorders), cathepsin L (metastases) or parasitic proteases such asfalcipain (malaria parasite Plasmodium falciparum), cruzipain(Trypanosoma cruzi infection). Recently a bacterial protease,staphylopain (S. aureus infection) has also been tentatively assigned toclan CA. X-ray crystallographic structures are available for a range ofthe above mentioned proteases in complex with a range of inhibitors e.g.papain (PDB entries, 1pad, 1pe6, 1pip, 1pop, 4pad, 5pad, 6pad, 1ppp,1the, 1csb, 1huc), cathepsin K (1au0, 1au2, 1au3, 1au4, 1atk, 1mem,1bgo, 1ayw, 1ayu), cathepsin L (1cs8), cathepsin S (currently on-hold,but published McGrath, M. E. et al, Protein Science, 7, 12941302, 1998),cruzain (a recombinant form of cruzipain see Ealdn, A. E. et al, 268(9),6115-6118, 1993) (1ewp, 1aim, 2aim, 1F29, 1F2A, 1F2B, 1F2C),staphylopain (1cv8). Each of the structures displays a similar overallactive-site topology, as would be expected by their ‘clan’ and ‘family’classification and such structural similarity exemplifies one aspect ofthe difficulties involved in discovering a selective inhibitor ofcruzipain suitable for human use. However, subtle differences in termsof the depth and intricate shape of the active site groove of each CA C1protease are evident, which may be exploited for selective inhibitordesign. Additionally, many of the current substrate-based inhibitorcomplexes of CA Cl family proteases show a series of conserved hydrogenbonds between the inhibitor and the protease backbone, which contributesignificantly to inhibitor potency. Primarily a bidentate hydrogen-bondis observed between the protease Gly66 (C═O)/inhibitor N—H and theprotease Gly66(NH)/inhibitor (C═O), where the inhibitor (C═O) and (NH)are provided by an amino acid residue NHCHRCO that constitutes the S2sub-site binding element within the inhibitor (see Berger, A. andSchecter, I. Philos. Trans. R. Soc. Lond. [Biol.], 257, 249-264, 1970for a description of protease binding site nomenclature). A furtherhydrogen-bond between the protease main-chain (C═O) of asparagine oraspartic acid (158 to 163, residue number varies between proteases) andan inhibitor (N—H) is often observed, where the inhibitor (N—H) isprovided by the S1 sub-site binding element within the inhibitor. Thus,the motif X—NHCHRCO—NH—Y is widely observed amongst the prior artsubstrate-based inhibitors of CA C1 proteases.

[0007] In the prior art, the development of cysteine protease inhibitorsfor human use has recently been an area of intense activity. Consideringthe CA C1 family members, particular emphasis has been placed upon thedevelopment of inhibitors of human cathepsins, primarily cathepsin K(osteoporosis), cathepsin S (autoimmune disorders) and cathepsin L(metastases), through the use of peptide and peptidomimetic nitriles(e.g. see WO-A-0109910, WO-A-0051998, WO-A-0119816, WO-A-9924460,WO-A-0049008, WO-A-0048992, WO-A-0049007, WO-A-0130772, WO-A-0055125,WO-A-0055126, WO-A-0119808, WO-A-0149288, WO-A-0147886), linear andcyclic peptide and peptidomimetic ketones (e.g. see Veber, D. F. andThompson, S. K., Curr. Opin. Drug Discovery Dev., 3(4), 362-369, 2000,WO-A-0170232, WO-A-0178734, WO-A-0009653, WO-A-0069855, WO-A-0029408,WO-A-0134153 to WO-A-0134160, WO-A-0029408, WO-A-9964399, WO-A-9805336,WO-A-9850533), ketoheterocycles (e.g. see WO-A-0055144, WO-A-0055124)and monobactams (e.g. see WO-A-0059881, WO-A-9948911, WO-A-0109169). Theprior art describes potent in vitro inhibitors, but also highlights themany difficulties in developing a human therapeutic. For example,WO-A-9850533 and WO-A-0029408 describe compounds that may be referred toas cyclic ketones and are inhibitors of cysteine proteases with aparticular reference towards papain family proteases and as a mostpreferred embodiment, cathepsin K. WO-A-9850533 describes compoundssubsequently detailed in the literature as potent inhibitors ofcathepsin K with good oral bioavailability (Witherington, J.,‘Tetrahydrofurans as Selective Cathepsin K Inhibitors’, RSC meeting,Burlington House, London, 1999). The compounds of WO-A-9850533 werereported to bind to cathepsin K through the formation of a reversiblecovalent bond between the tetrahydrofuran carbonyl and the active sitecatalytic cysteine residue (Witherington, J., 1999). Additionally, thesame cyclic ketone compounds are described in WO-A-9953039 as part of awide-ranging description of inhibitors of cysteine proteases associatedwith parasitic diseases, with particular reference to the treatment ofmalaria by inhibition of falcipain. However, subsequent literaturedescribes the cyclic ketone compounds of WO-A-9850533 to be unsuitablefor further development or for full pharmacokinetic evaluation due to aphysiochemical property of the inhibitors, the poor chiral stability ofthe α-aminoketone chiral centre (Marquis, R. W. et al, J. Med. Chem.,44(5), 725-736, 2001). WO-A-0069855 describes compounds that may also bereferred to as cyclic ketones with particular reference towardsinhibition of cathepsin S. The compounds of WO-A-0069855 are consideredto be an advance on compounds of WO-A-9850533 due to the presence of theβ-substituent on the cyclic ketone ring system that provides chiralstability to the α-carbon of the cyclic ketone ring system. However, thecompounds of WO-A-0069855 and indeed those of WO-A-9850533 describe anabsolute requirement for the presence of an amino acid substituent R¹R²NHCHR³ CO— that provides the N-terminal portion of the inhibitormolecules, i.e. contain the potential hydrogen-bonding motifX—NHCHRCO—NH—Y that is widely observed amongst the prior artsubstrate-based inhibitors of CA C1 proteases. Although the number ofamino acids described both in the literature and commercially availableconstitute many hundreds of possibilities, in many instances an aminoacid backbone may not provide the appropriate balance of propertiesrequired for further inhibitor development.

[0008] It has now been discovered that certain compounds, defined bygeneral formula (I), are potent and selective cruzipain proteaseinhibitors which are useful in the treatment of Trypanosoma cruziinfection. Other compounds defined by general formula (I) are proteaseinhibitors across a broad range of CA C1 cysteine proteases andcompounds useful in the treatment of diseases caused by cysteineproteases. Compounds described by general formula (I) do not contain theX—NHCHRCO—NH—Y motif that is widely observed amongst the prior artsubstrate-based inhibitors of CA C1 proteases, yet surprisinglycompounds defined by general formula (I) retain good potency. Thepresent invention provides substituted(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide,(2-alkyl-4-oxo-tetrahydrothiophen-3-yl) amide and(2-alkyl-5-oxocyclopentyl)amide compounds defined by general formula(I).

[0009] Accordingly, the first aspect of the invention provides acompound according to general formula (I):

[0010] wherein:

[0011] R¹=C₀₋₇-alkyl (when C=0, R¹ is simply hydrogen), C₃₋₆-cycloalkylor Ar—C₀₋₇-alkyl (when C=0, R¹ is simply an aromatic moiety Ar);

[0012] R²=C₁₋₇-alkyl, C₃₋₆-cycloalkyl or Ar—C₀₋₇-alkyl;

[0013] Y=CHR³—CO or CR³R⁴—CO where R³ and R⁴ are independently chosenfrom C₀₋₇-alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl, or Y represents

[0014] where L is a number from one to four and R⁵ and R⁶ areindependently chosen from CR⁷R⁸ where R⁷ and R⁸ are independently chosenfrom C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl or halogen; and for eachR⁵ and R⁶ either R⁷ or R⁸ (but not both R⁷ and R⁸) may additionally bechosen from O—C₀₋₇-alkyl, O—C₃₋₆-cycloalkyl, O—Ar—C₀₋₇-alkyl,S—C₀₋₇-alkyl, S—C₃₋₆-cycloalkyl, S—Ar—C₀₋₇-alkyl, NH—C₀₋₇-alkyl,NH—C₃₋₆-cycloalkyl, NH—Ar—C₀₋₇-alkyl, N—(C₀₋₇-alkyl)₂,N—(C₃₋₆-cycloalkyl)₂, and N—(Ar—C₀₋₇-alkyl)₂;

[0015] (X)_(o)=CR⁹R¹⁰, where R⁹ and R¹⁰ are independently chosen fromC₀₋₇-alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl and o is a number fromzero to three;

[0016] (W)_(n)=O, S, C(O), S(O) or S(O)₂ or, when o is one or greater,NR¹¹, where R¹¹ is chosen from C₀₋₇-alkyl, C₃₋₆-cycloalkyl andAr—C₀₋₇-alkyl and n is zero or one;

[0017] (V)_(m)=C(O), C(S), S(O), S(O)₂, S(O)₂NH, OC(O), NHC(O), NHS(O),NHS(O)₂, OC(O)NH, C(O)NH or CR¹² R¹³, where R¹² and R¹³ areindependently chosen from C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl andm is a number from zero to three, provided that when m is greater thanone, (V)_(m) contains a maximum of one carbonyl or sulphonyl group;

[0018] Z=O (in which case compounds of general formula (I) may be namedas (2-alkyl-4-oxo-tetrahydrofuran-3-yl)amides),

[0019] S (in which case compounds of general formula (I) may be named as(2-alkyl-4-oxo-tetrahydrothiophen-3-yl)amides), or

[0020] CH₂ (in which case compounds of general formula (I) may be namedas (2-alkyl-5-oxocyclopentyl)amides);

[0021] U=a stable 5- to 7-membered monocyclic or a stable 8- to11-membered bicyclic ring which is either saturated or unsaturated andwhich includes zero to four heteroatoms (as detailed below):

[0022] wherein R¹⁴ is chosen from:

[0023] C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl, halogen,O—C₀₋₇-alkyl, O—C₃₋₆-cycloalkyl, O—Ar—C₀₋₇-alkyl, S—C₀₋₇-alkyl,S—C₃₋₄-cycloalkyl, S—Ar—C₇-ayl, NH—CO-7-alkyl, NH—C₃₋₆-cycloalkyl,NH—Ar—C₀₋₇-alkyl, N—(C₀₋₇-alkyl)₂, N—(C₃₋₆-cycloalkyl)₂ andN—(Ar—C₀₋₇-alkyl)₂;

[0024] A is chosen from:

[0025] CH₂, CHR¹⁴, O, S and NR¹⁵, where R¹⁴ is as defined above andwhere R¹⁵ is chosen from C₀₋₇alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl;

[0026] B, D and G are independently chosen from:

[0027] CR¹⁴, where R¹⁴ is as defined above, or N;

[0028] E is chosen from:

[0029] CH₂, CHR¹⁴, O, S and NR¹⁵, where R¹⁴ and R¹⁵ are defined asabove;

[0030] J, L, M, R, T, T₂, T₃ and T₄ are independently chosen from:

[0031] CR¹⁴ and N, where R¹⁴ is as defined above;

[0032] T₅ is chosen from:

[0033] CH or, only when m+n+o≧1, T₅ may additionally be N;

[0034] q is a number from one to three, thereby defining a 5-, 6- or7-membered ring.

[0035] B, D, G, J, L, M, R, T, T₂, T₃ and T₄ may additionally representan N-oxide (N→O).

[0036] The present invention includes all salts, hydrates, solvates,complexes and prodrugs of the compounds of this invention. The term“compound” is intended to include all such salts, hydrates, solvates,complexes and prodrugs, unless the context requires otherwise.

[0037] Appropriate pharmaceutically and veterinarily acceptable salts ofthe compounds of general formula (I) include salts of organic acids,especially carboxylic acids, including but not limited to acetate,trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate,digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate,heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate,3-phenylpropionate, picrate, pivalate, proprionate, tartrate,lactobionate, pivolate, camphorate, undecanoate and succinate, organicsulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids. Salts which are not pharmaceutically orveterinarily acceptable may still be valuable as intermediates.

[0038] Prodrugs are any covalently bonded compounds which release theactive parent drug according to general formula (I) in vivo. A prodrugmay for example constitute an acetal or hemiacetal derivative of theexocyclic ketone functionality present in the(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide,(2-alkyl-4-oxo-tetrahydrothiophen-3-yl) amide and(2-alkyl-5-oxocyclopentyl)amide scaffold. If a chiral centre or anotherform of isomeric centre is present in a compound of the presentinvention, all forms of such isomer or isomers, including enantiomersand diastereoisomers, are intended to be covered herein. Compounds ofthe invention containing a chiral centre may be used as a racemicmixture, an enantiomerically enriched mixture, or the racemic mixturemay be separated using well-known techniques and an individualenantiomer may be used alone.

[0039] ‘Halogen’ as applied herein is meant to include F, Cl, Br, I;

[0040] ‘Heteroatom’ as applied herein is meant to include O, S and N;

[0041] ‘C₀₋₇-alkyl’ as applied herein is meant to include stablestraight and branched chain aliphatic carbon chains containing zero(i.e. simply hydrogen) to seven carbon atoms such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, isopentyl,hexyl, heptyl and any simple isomers thereof. Additionally, anyC₀₋₇-alkyl may optionally be substituted at any point by one, two orthree halogen atoms (as defined above) for example to give atrifluoromethyl substituent. Furthermore, C₀₋₇-alkyl may contain one ormore heteroatoms (as defined above) for example to give ethers,thioethers, sulphones, sulphonamides, substituted amines, amidines,guanidines, carboxylic acids, carboxamides. If the heteroatom is locatedat a chain terminus then it is appropriately substituted with one or twohydrogen atoms. A heteroatom or halogen is only present when C₀₋₇-alkylcontains a minimum of one carbon atom.

[0042] C₁₋₇-alkyl as applied herein is meant to include the definitionsfor C₀₋₇-alkyl (as defined above) but describes a substituent thatcomprises a minimum of one carbon.

[0043] ‘C₃₋₆-cycloalkyl’ as applied herein is meant to include anyvariation of ‘C₀₋₇-alkyl’ which additionally contains a carbocyclic ringsuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. Thecarbocyclic ring may optionally be substituted with one or more halogens(as defined above) or heteroatoms (as defined above) for example to givea tetrahydrofuran, pyrrolidine, piperidine, piperazine or morpholinesubstituent.

[0044] ‘Ar—C₀₋₇-alkyl’ as applied herein is meant to include anyvariation of C₀₋₇-alkyl which additionally contains an aromatic ringmoiety ‘Ar’. The aromatic ring moiety Ar can be a stable 5 or 6-memberedmonocyclic or a stable 9 or 10 membered bicyclic ring which isunsaturated, as defined previously for U in general formula (I). Thearomatic ring moiety Ar may be substituted by R¹⁴ (as defined above forU in general formula (I)). When C=0 in the substituent Ar—C₀₋₇-alkyl,the substituent is simply the aromatic ring moiety Ar.

[0045] Other expressions containing terms such as alkyl and cycloalkylare intended to be construed according to the definitions above. Forexample “C₁₋₄ alkyl” is the same as C₀₋₇-alkyl except that it containsfrom one to four carbon atoms.

[0046] If different structural isomers are present, and/or one or morechiral centres are present, all isomeric forms are intended to becovered. Enantiomers are characterised by the absolute configuration oftheir chiral centres and described by the R- and S-sequencing rules ofCahn, Ingold and Prelog. Such conventions are well known in the art(e.g. see ‘Advanced Organic Chemistry’, 3^(rd) edition, ed. March, J.,John Wiley and Sons, New York, 1985).

[0047] Preferred compounds of general formula (I) include those in whichR¹ comprises C₀₋₇-alkyl or Ar—C₀₋₇-alkyl. Thus, for example, preferredR¹ moieties include hydrogen, or a straight or branched alkyl chain, ora straight or branched heteroalkyl chain, or an optionally substitutedarylalkyl chain, or an optionally substituted arylheteroalkyl chain.

[0048] It is particularly preferred that R¹ is hydrogen or C₁₋₄ alkyl orAr—C₁₋₄-alkyl and examples of such R¹ substituents include, but are notlimited to:

[0049] It is preferred that R² is C₁₋₇-alkyl or Ar—C₀₋₇-alkyl, forexample, straight or branched alkyl chains or heteroalkyl chains oroptionally substituted aralkyl chains or an alkylcarboxylic ester chainor an N-(alkylcarbonyl)sulphonamide chain or an alkyl carboxamide chain.

[0050] Particularly preferred compounds include those in which R² isC₁₋₄-alkyl or Ar—C₁₋₄-alkyl and examples of such R² substituentsinclude, but are not limited to:

[0051] wherein R¹⁴ and R¹⁵ are as defined previously.

[0052] Additionally, preferred compounds of general formula (I) comprisethose in which Z=‘O’.

[0053] Combining the preferred examples of the substituents R¹ and R²with an exemplary (2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide scaffoldyields the following compounds, which are among those preferred:

[0054] In preferred compounds of general formula (I), Y is CHR⁴CO whereR⁴ is selected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl, for example hydrogen, astraight or branched alkyl chain, a straight or branched heteroalkylchain, an optionally substituted arylalkyl chain or an optionallysubstituted arylheteroalkyl chain. Additionally in preferred compoundsof general formula (I), R⁴ is selected from C₃₋₆-cycloalkyl, for examplecyclohexylmethyl or cyclopentylmethyl.

[0055] Other preferred compounds of general formula (I) are those inwhich Y comprises a group:

[0056] where R⁵ and R⁶ are each CR⁷R⁸ and each R⁷ and R⁸ is,independently, selected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl, for examplehydrogen, a straight or branched alkyl chain, a straight or branchedheteroalkyl chain, an optionally substituted arylalkyl chain or anoptionally substituted arylheteroalkyl chain.

[0057] More preferred R⁷ and R⁸ groups include C₀₋₄ alkyl, for examplehydrogen, methyl and ethyl.

[0058] Examples of preferred Y substituents include the following:

[0059] wherein R¹⁴ and R¹⁵ and Ar are as defined previously.

[0060] More preferred compounds of general formula (I), comprise an R⁴group chosen from C₁₋₄-alkyl, which may be substituted with OH, NR¹⁵R¹⁵,COOR¹⁵, or CONR¹⁵; or Ar—C₁₋₄-alkyl, where the aryl group may besubstituted with R¹⁴; wherein each R¹⁴ and R¹⁵ is independently asdefined above.

[0061] Even more preferred R⁴ groups comprise Ar—CH₂—, where thearomatic ring is an optionally substituted phenyl or monocyclicheterocycle Additionally, even more preferred R⁴ groups comprise simplebranched alkyl groups such as isobutyl or straight heteroalkyl chainssuch as benzylsulfanylmethyl or benzylsulphonylmethyl. Furthermore, evenmore preferred R⁴ groups comprise cyclohexylmethyl. Examples of evenmore preferred Y substituents comprise the following,

[0062] wherein R¹⁴ and Ar are as defined previously

[0063] It is preferred that in the group (X)_(o), each of R⁹ and R¹⁰ isselected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl, for example hydrogen, astraight or branched alkyl chain, a straight or branched heteroalkylchain, an optionally substituted arylalkyl chain or an optionallysubstituted arylheteroalkyl chain.

[0064] More preferred (X)_(o) groups comprise R⁹ chosen from hydrogen;R¹⁰ is C₁₋₄-alkyl, which may be substituted with OH, NR¹⁵R¹⁵, COOR¹⁵, orCONR¹⁵; or Ar—C₁₋₄-alkyl, where the aryl group may be substituted withR¹⁴, wherein each R¹⁴ and R¹⁵ is independently as defined above.

[0065] Examples of preferred (X)_(o) groups include the following:

[0066] wherein R¹⁴ and R¹⁵ are as defined previously.

[0067] Even more preferred compounds of general formula (I), comprise(X)O groups that are simple alkyl groups such as methylene and where o=0or 1.

[0068] In the group (W)_(n):

[0069] W is preferably O, S, SO₂, S(O), C(O) or when o is one orgreater, NR¹¹, where R¹¹ is C₀₋₄-alkyl; and n is 0 or 1.

[0070] More preferred compounds of general formula (I), comprise (W)_(n)groups defined as O, S, SO₂, C(O) and where n=0 or 1.

[0071] In the group (V)_(m):

[0072] V is preferably C(O), C(O)NH or CHR¹³, where R¹³ is C₀₋₄-allyl;and m is 0 or 1.

[0073] Preferred V and W substituent combinations encompassed by generalformula (I) include, but are not limited to:

[0074] More preferred V, W and X substituent combinations encompassed bygeneral formula (I) comprise, but are not limited to

[0075] In preferred compounds of general formula (I), U comprises anoptionally substituted 5- or 6-membered saturated or unsaturatedheterocycle or Ar group or an optionally substituted saturated orunsaturated 9- or 10-membered heterocycle or Ar group. Examples of suchpreferred U rings include the following:

[0076] and also the following

[0077] wherein R¹⁴ is as defined previously.

[0078] More preferred compounds of general formula (I), contain a Ugroup comprising of a bulky alkyl or aryl group at the para position ofan aryl Ar. Also, more preferred compounds contain a meta or para-biarylAr—Ar, where Ar is as previously defined. Additionally, more preferredcompounds contain a 6, 6 or 6, 5 or 5,6-fused aromatic ring. Examples ofmore preferred U groups are

[0079] wherein R¹⁴, D, E, G. J. L, M, R, T, T₂, T₃ and T₄ are as definedpreviously.

[0080] Even more preferred compounds of general formula (I),particularly for inhibition of cruzipain, contain a U group comprising a6-membered Ar ring containing a bulky alkyl or aryl group at the paraposition, where Ar is as previously defined

[0081] wherein R¹⁴, D, E, G, J, L, M, R and T are as defined previously

[0082] Yet even more preferred compounds of general formula (I), containa U group comprising but are not limited to the following,

[0083] wherein R¹⁴, D, E, G, J and L are as defined previously.

[0084] Abbreviations and symbols commonly used in the peptide andchemical arts are used herein to describe compounds of the presentinvention, following the general guidelines presented by the IUPAC-IUBJoint Commission on Biochemical Nomenclature as described in Eur. J.Biochem., 158, 9-, 1984. Compounds of formula (I) and the intermediatesand starting materials used in their preparation are named in accordancewith the IUPAC rules of nomenclature in which the characteristic groupshave decreasing priority for citation as the principle group. An examplecompound of formula (I), compound (1) in which R¹ is H, R² is methyl, Zis oxygen, Y is 4-methylpentyl, (X)_(o) is zero, (W)_(n) is oxygen,(v)_(m) is methylene and U is phenyl is thus named:—

(1) 2S-Benzyloxy-4-methyl-pentanoic acid(2R-methyl-4-oxo-tetrahydrofuran-3S-yl)-amide

[0085] A second example compound of formula (I), compound (2) in whichR¹ is H, R² is methyl, Z is sulphur, Y is 4-methylpentyl, (X)₀ is zero,(W)_(n) is oxygen, (V)_(m) is methylene and U is phenyl is thus named:—

[0086] 2S-Benzyloxy-4-methyl-pentanoic Acid(2R-methyl-4-oxo-tetrahydrothiophen-3R-yl)amide

[0087] A third example compound of formula (I), compound (3) in which R¹is H, R² is methyl, Z is methylene, Y is 4-methylpentyl, (X)₀ is zero,(W)_(n) is oxygen, (V)_(m) is methylene and U is phenyl is thus named:—

2S-Benzyloxy-4-methylpentanoic Acid(2S-methyl-5-oxo-cyclopent-3S-yl)amide

[0088] Compounds of the invention include, but are not limited to, thefollowing examples where all 4 stereoisomeric combinations of the cyclicketone are included, i.e. (2S, 3S), (2R, 3S), (2S, 3R), (2R, 3R) andwhere Z=‘O’ and R¹=‘H’, and also include the equivalent analoguesincluded in the full definition of Z and R¹ and R²

[0089] 4-Methyl-2-(4-trifluoromethylbenzyloxy)pentanoic acid (2-methyl4-oxo-tetrahydro furan-3-yl)-amide

[0090]N-(2-Methyl-4-oxo-tetrahydro-furan-3-yl)-3-phenyl-2-(4-trifuoromethylbenzyloxy)-propionamide

[0091]3-(4-Hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(4-trifluoromethyl-benzyloxy)-propionamide

[0092] 2-Benzyloxy-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0093]2-Benzyloxy-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-3-phenyl-propionamide

[0094]2-Benzyloxy-3-(4-hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0095] 4-Methyl-2-(4-thiophen-2-yl-benzyloxy)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0096]N-(2-Methyl-4-oxo-tetrahydro-furan-3-yl)-3-phenyl-2-(4-thiophen-2-yl-benzyloxy)-propionamide

[0097]3-(4-Hydroxyphenyl)-N-(2-methyl-oxo-4-tetrahydro-furan-3-yl)-2-(4-thiophen-2-yl-benzyloxy)-propionamide

[0098] 2-(4-tert-Butyl-benzyloxy)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0099]2-(4-tert-Butyl-benzyloxy)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-3-phenyl-propionamide

[0100]2-(4-tert-Butyl-benzyloxy)-3-(4-hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0101] 2-(Biphenylyl-4-methoxy)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0102]2-(Biphenyl-4-ylmethoxy)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-3-phenyl-propionamide

[0103]2-(Biphenyl-4-ylmethoxy)-3-(4-hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0104] 2-(4-tert-Butyl-benzylsulfanyl)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0105]2-(4-tert-Butyl-benzylsulfanyl)-3-(4-hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide2-(Biphenyl-4-ylmethylsulfanyl)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0106]2-(Biphenyl-4-ylmethylsulfanyl)-3-(4-hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0107] 4-Methyl-2-(4-thiophen-2-yl-benzylsulfanyl)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0108]3-(4-Hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(4-thiophen-2-yl-benzylsulfanyl)-propionamide

[0109] 2-(4-tert-Butyl-phenylmethanesulfonyl)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0110]2-(4-tert-Butyl-phenylmethanesulfonyl)-3-(4-hydroxyphenyl)-N(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0111] 2-(Biphenyl-4-ylmethanesulfonyl)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0112]2-(Biphenyl-4-ylmethanesulfonyl)-3-(4-hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0113] 4-Methyl-2-(4-thiophen-2-yl-phenylmethanesulfonyl)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0114]3-(4-Hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(4-thiophen-2-yl-phenylmethanesulfonyl)-propionamide

[0115] 3-Phenyl-pyrrole-1-carboxylic acid2-(4-hydroxyphenyl)-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylester

[0116] 3-Phenyl-pyrrole-1-carboxylic acid3-methyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-butyl ester

[0117] 1,3-Dihydro-isoindole-2-carboxylic acid2-(4-hydroxyphenyl)-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylester

[0118] 1,3-Dihydro-isoindole-2-carboxylic acid3-methyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-butyl ester

[0119] 3,4-Dihydro-1H-isoquinoline-2-carboxylic acid2-(4-hydroxyphenyl)-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylester

[0120] 3,4-Dihydro-1H-isoquinoline-2-carboxylic acid3-methyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-butyl ester

[0121]2-(4-Hydroxybenzyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-4-oxo-4-(3-phenylpyrrol-1-yl)-butyramide

[0122] 4-Methyl-2-[2-oxo-2-(3-phenyl-pyrrol-1-yl)-ethyl]-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0123]4-(1,3-Dihydro-isoindol-2-yl)-2-(4-hydroxybenzyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-4-oxo-butyramide

[0124] 2-[2-(1,3-Dihydro-isoindol-2-yl)-2-oxo-ethyl]4-methyl-pentanoicacid (2-methyl-4 oxo-tetrahydro-furan-3-yl)-amide

[0125]4-(3,4-Dihydro-1H-isoquinolin-2-yl)-2-(4-hydroxybenzyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-4-oxo-butyramide

[0126]2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]-4-methyl-pentanoicacid (2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0127] Additional compounds of the invention include, but are notlimited to, the following examples where all 4 stereoisomericcombinations of the cyclic ketone are included, i.e. (2S, 3S), (2R, 3S),(2S, 3R), (2R, 3R) and where Z=‘O’ and R¹=‘H’, and also include theequivalent analogues included in the full definition of Z and R′ and R²

[0128]3-(4-Hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(naphthalen-1-ylmethoxy)-propionamide

[0129]N-(2-Methyl-4-oxo-tetrahydro-furan-3-yl)-2-(naphthalen-1-ylmethoxy)-3-phenyl-propionamide

[0130] 2-Benzyloxy-3-cyclohexyl-N-(2-methyloxo-tetrahydro-furan-3-yl)-propionamide

[0131]3-Cyclohexyl-2-(furan-3-ylmethoxy)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0132]3-Cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(thiophen-3-ylmethoxy)-propionamide

[0133]3-Cyclohexyl-2-(furan-2-ylmethoxy)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0134]3-Cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(thiophen-2-ylmethoxy)-propionamide

[0135]2-(Benzo[b]thiophen-3-ylmethoxy)-3-cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0136] 2-(Furan-3-ylmethoxy)-4,4-dimethyl-pentanoic acid(2-methyl-1-oxo-tetrahydro-furan-3-yl)-amide

[0137] 4,4-Dimethyl-2-(thiophen-3-ylmethoxy)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0138] 2-(Furan-2-ylmethoxy)-4,4-dimethyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)amide

[0139] 4,4-Dimethyl-2-(thiophen-2-ylmethoxy)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0140] 2-(Benzo[b]thiophen-3-ylmethoxy)-4,4-dimethyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0141]3-Cyclohexyl-2-(furan-3-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0142]3-Cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(thiophen-3-ylmethylsulfanyl)-propionamide

[0143]3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydro-furan-2-yl)-propionamide

[0144]3-Cyclohexyl-N-(2-methyl-1-oxo-tetrahydro-furan-3-yl)-2-(thiophen-2-ylmethylsulfanyl)-propionamide

[0145]2-(Benzo[b]thiophen-3-ylmethylsulfanyl)-3-cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0146] 2-(Furan-3-ylmethylsulfanyl)-4,4 dimethyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0147] 4,4-Dimethyl-2-(thiophen-3-ylmethylsulfanyl)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0148] 2-(Furan-2-ylmethylsulfanyl)-4,4-dimethyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0149] 4,4-Dimethyl-2-(thiophen-2-ylmethylsulfanyl)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0150] 2-(Benzo[b]thiophen-3-ylmethylsulfanyl)-4-dimethyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0151]3-(4-Hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-naphthalen-1-ylmethylsulfanyl)-propionamide

[0152]3-Cyclohexyl-2-(furan-3-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0153]3-Cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(thiophen-3-ylmethanesulfonyl)-propionamide

[0154]3-Cyclohexyl-2-(furan-2-ylmethylsulfonyl)-N-(2-methyl-4-oxo-tetrahydro-furan-2-yl)-propionamide

[0155]3-Cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(thiophen-2-ylmethylsulfonyl)-propionamide

[0156]2-(Benzo[b]thiophen-3-ylmethylsulfonyl)-3-cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0157] 2-(Furan-3-ylmethylsulfonyl)-4,4-dimethyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0158] 4,4-Dimethyl-2-(thiophen-3-ylmethylsulfonyl)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0159] 2-(Furan-2-ylmethylsulfonyl)-4,4-dimethyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0160] 4,4-Dimethyl-2-(thiophen-2-ylmethylsulfonyl)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0161] 2-(Benzo[b]thiophen-3-ylmethylsulfonyl)-4,4-dimethyl-pentanoicacid (2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0162]3-(4-Hydroxyphenyl)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-2-(naphthalen-1-ylmethylsulfonyl)-propionamide

[0163] Morpholine-4-carboxylic acid2-cyclohexyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylester

[0164] Morpholine4-carboxylic acid3,3-dimethyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-butylester

[0165]2-Cyclohexylmethyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-4-morpholin-4-yl-4-oxo-butyramide

[0166] 4,4-Dimethyl-2-(2-morpholinyl-2-oxo-ethyl)-pentanoic acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0167] 2-Biphenyl-3-yl-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydro-fan-3-yl)-amide

[0168] To those skilled in the practices of organic chemistry, compoundsof general formula (I) may be readily synthesised by a number ofchemical strategies, performed either in solution or on the solid phase(see Atherton, E. and Sheppard, R. C. In ‘Solid Phase Peptide Synthesis:A Practical Approach’, Oxford University Press, Oxford, U.K. 1989, for ageneral review of solid phase synthesis principles). The solid phasestrategy is attractive in being able to generate many thousands ofanalogues, typically on a 5-100 mg scale, through established parallelsynthesis methodologies (e.g. see (a) Bastos, M.; Maeji, N.J.; Abeles,R. H. Proc. Natl. Acad. Sci. USA, 92, 6738-6742, 1995).

[0169] Therefore, one strategy for the synthesis of compounds of generalformula (I) comprises:—

[0170] (a) Preparation of an appropriately functionalised and protected(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide or a(2-alkyl-4-oxo-tetrahydrothiophen-3-yl)amide or a(2-alkyl-5-oxocyclopentyl)amide building block in solution.

[0171] (b) Attachment of the building block (a) to the solid phasethrough a linker that is stable to the conditions of synthesis butreadily labile to cleavage at the end of a synthesis (see James, I. W.,Tetrahedron, 55(Report N° 489), 4855-4946, 1999, for examples of the‘linker’ function as applied to solid phase synthesis).

[0172] (c) Solid phase organic chemistry (see Brown, P D. J. Chem. Soc.,Perkin Trans. 1, 19, 3293-3320, 1998), to construct the remainder of themolecule.

[0173] (d) Compound cleavage from the solid phase into solution.

[0174] (e) Cleavage work-up and compound analysis.

[0175] The first stage in a synthesis of compounds of general formula(I) is the preparation in solution of a functionalised and protectedbuilding block. A typical scheme towards the(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide (6) is detailed in Scheme 1.

[0176] Scheme 1. (a) ¹BuOCOCl, NMM, DCM, −15° C., 10 mins, under argon.(b) Diazomethane in diethyl ether, −15° C. to RT over 1 hr. (c) Aceticacid (d) LiCl (10 eq) in 80% aq acetic acid, 5° C. to RT over 1 hr.

[0177] FmOC(O) denotes the well known amine protecting group 9-fluorenylmethoxycamonyl (Fmoc, see Atherton, E. and Sheppard, R. C., 1989) and‘Pg’ denotes either a free hydroxyl or an hydroxylprotecting group suchas tert-butyl ether. In the illustrated case, condensation withdiazomethane provides R¹=H.

[0178] Considering step (a), synthesis may commence from suitablyprotected β-hydroxy-α-amino acids (4), which are accessible through avariety of literature methods e.g. (a) Adams, Z. M., Jackson, R. F. W.,Palmer, N.J., Rami, H. K, Wythes, M. J., J. Chem. Soc., Perkin Trans I,937 -947, 1999, (b) Hubschwerlen, C., et al, J. Med. Chem., 4,3972-3975, 1998, (c) Luzzio, F. A., et al, Tet. Lett., 41, 7151-7155,2000, (d) Morgan, A. J. et al, Org. Lett. 1(12), 1949-1952, 1999. (e)Zhang, H., Xia, P., Zhou, W., Tetrahedron: Asymmetry, 11, 3439-3447,2000, (f) Blaskovich, M. A., et al, J. Org. Chem., 6, 3631-3646, 1998.

[0179] In the simplest case where R² is a methyl substituent, theβ-hydroxy-α-amino acid (4) is threonine (Thr) for which all fourstereosiomers (i.e. α ‘R’ or ‘S’ and β ‘R’ or ‘S’) are commerciallyavailable. Activation of the suitably protected β-hydroxy-α-amino acids(4) via isobutyl chloroformate mixed anhydride, followed by condensationwith diazomethane, yields the diazomethylketone intermediates (5).Treatment of diazomethylketone intermediates (5) with lithium chloridein aqueous acetic acid provides the protected(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide (6). Introduction of simple R¹substituents may be achieved by condensation of activated (4) withalternatives to diazomethane such as diazoethane (R¹═CH₃), or1-phenyloxydiazoethane (R¹═CH₂OPh).

[0180] The protected building blocks (synthesis exemplified by the(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide (6) detailed in Scheme 1) maybe utilised in a solid phase synthesis of inhibitor molecules (steps (b)to (e)). Step (b), the solid phase linkage of an aldehyde or ketone, haspreviously been described by a variety of methods (e.g. see (a) James,I. W., 1999, (b) Lee, A., Huang, L., Ellman, J. A., J. Am. Chem. Soc,121(43), 9907-9914, 1999, (c) Murphy, A. M., et al, J. Am. Chem. Soc,114, 3156-3157, 1992). A suitable method amenable to the reversiblelinkage of an alkyl ketone functionality such as (6) is through acombination of the previously described chemistries. The semicarbazide,4-[[(hydrazinocarbonyl)amino] methyl]cyclohexane carboxylicacid.trifluoroacetate (7) (Murphy, A. M., et al, J. Am. Chem. Soc, 114,3156-3157, 1992), may be utilised as illustrated in Scheme 2,exemplified by linkage of the (2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide(6).

[0181] Scheme 2. (a) (6) in 90% EtOH/H₂O 1.5 eqNaOAc/4-[[(hydrazinocarbonyl)amino] methyl]cyclohexane carboxylicacidtrifiuoroacetate (7), 2 hr reflux. (O) 3 eq construct(8)/HBTU/HOBt/NMM, NH₂-SOLID PHASE, DMF, RT, o/n. (c) 20%piperidine/DMF, 30 mins. (d) Range of chemistries to introduce U-V-W-X-Y(e) TFA/H₂O (95:5, v/v), RT, 2 hr.

[0182] Construct (8) is prepared through reaction of the linker molecule(7) and the (2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide (6) by reflux inaqueous ethanol/sodium acetate. Standard solid phase techniques (e.g.see Atherton, E. and Sheppard, R. C., 1989) are used to anchor theconstruct to an amino-functionalised solid phase through the freecarboxylic acid functionality of (8), providing the loaded construct(9).

[0183] Loaded construct (9) may be reacted with a wide range ofcarboxylic acids available commercially or in the literature, tointroduce the left-hand portion ‘U-V-W-X-Y’ in general formula (I). Inthe simplest example, the entire left hand portion of an inhibitor ofgeneral formula (I) can be prepared in solution by traditional organicchemistry methods and coupled to construct (9) on the solid phase(Schemes 3-7). For example (Scheme 3), treatment in solution of an aminoacid, exemplified by (10) with sodium nitrite/H₂SO₄, provides theα-hydroxyacid, exemplified by (11) (Degerbeck, F. et al, J. Chem. Soc,Perkin Trans. 1, 11-14, 1993). Treatment of α-hydroxyacid, (11) withsodium hydride in a dimethylformamide/dichloromethane mixture followedby addition of benzyl bromide, provides2RS-benzyloxy-3-cyclohexylpropionic acid (12). Coupling of (12) to thesolid phase construct (9) followed by cleavage, provides (13), anexample of general formula (I) where R³=‘H’, (X)₀=‘-’, (W)_(n)=‘O’, n=1,(V)_(m)=‘CH₂’, i.e. R¹², R¹³=‘H’, m=1 and U=phenyl. To those skilled inthe practices of organic synthesis, a wide variety of aminoacids such as(10) may be converted to the corresponding α-hydroxyacid such as (11)following the general conditions detailed. Additionally, benzylbromidemay be replaced by any reasonable Ar—CR¹²R¹³-halogen, providing manyvariations of carboxylic acid (12) following the general conditionsdetailed. In certain instances, it may be advantageous to temporarilyprotect the carboxylic acid as the methyl ester (for example compound(18), Scheme 5) prior to reaction with the alkylhalide. The esterintermediate is then simply hydrolysed to acid (12). Thus analogues of(13) exploring a wide range of (V)_(m) and U in general formula (I) maybe prepared through the general conditions detailed in Scheme 3. Sincethe final synthetic step involves a trifluoroacetic acid (TFA) mediatedcleavage of the solid phase bound compound, compounds where thesubstituted ether is labile to TFA may be prepared in solution by analternative route (see Scheme 10).

[0184] Alternatively, coupling of construct (9) (following removal ofFmoc) with the α-hydroxyacid (11), provides a versatile solid phasebound intermediate ‘Y’ substituent in general formula (I) that may bereacted with many reagents. For example, the α-hydroxyl can be reactedunder Mitsunobu conditions (Hughes, D. L. Org. React. (N.Y), 42,335-656, 1992) to give ethers (i.e. X=‘-’, W=‘O’, in general formula(I)) (see Grabowska, U. et al, J. Comb. Chem., 2(5), 475490, 2000, foran example of Mitsunobu reaction on the solid phase). Alternatively, theα-hydroxyl can be reacted with a carbamoyl chloride to give a carbamate(i.e. X=‘-’, W=‘O’, V=‘NHC(O)’, in general formula (I)).

[0185] Alternatively, (Scheme 4), treatment in solution of an aminoacid, exemplified by (10) with sodium nitrite/H₂SO₄/potassium bromideprovides the α-bromoacid, exemplified by (14) (Souers, A. J. et al,Synthesis, 4, 583-585, 1999) with retention of configuration. Treatmentof α-bromoacid (14) with an alkylthiol exemplified byfuran-2-ylmethanethiol (15) in dimethylformamide/triethylamine, provides3-cyclohexyl-2R-(furan-2-ylsulfanyl)propionic acid (16), with inversionof configuration. Coupling of (16) to the solid phase construct (9)followed by cleavage, provides (17), an example of general formula (I)where R³=‘H’, (X)_(o)=‘-’, (W)_(n)=‘S’, n=1, (V)_(m)=‘CH₂’, i.e. R¹²,R¹³=‘H’, m=1 and U=2-furanyl. To those skilled in the practices oforganic synthesis, a wide variety of aminoacids such as (10) may beconverted to the corresponding α-bromoacid such as (14) following thegeneral conditions detailed. Additionally, furan-2-ylmethanethiol (15)may be replaced by any reasonable Ar—CR¹²R¹³-SH, providing manyvariations of carboxylic acid (16) following the general conditionsdetailed. Thus analogues of (17) exploring a wide range of (V)_(m) and Uin general formula (I) may be prepared through the general conditionsdetailed in Scheme 4.

[0186] Alternatively, coupling of construct (9) (following removal ofFmoc) with an α-bromoacid e.g. (14), provides a versatile intermediate‘Y’ substituent in general formula (I) that may be reacted with manyreagents. For example, the α-bromide can be displaced with nucleophilese.g. alcohols, thiols, carbanions etc, to give ethers (i.e. X=‘-’,W=‘O’, in general formula (I)), thioethers (i.e. X=‘-’, W=‘S’, ingeneral formula (I)). The thioethers may optionally be oxidised to thesulphone (see Scheme 8, i.e. X=‘-’, W=‘SO₂’, in general formula (I))(see Grabowska, U. et al, J. Comb. Chem., 2(5), 475-490, 2000, for anexample of bromide displacement and thioether oxidation on the solidphase).

[0187] Alternatively, (Scheme 5), treatment of an α-hydroxyacid,exemplified by (11) with trimethylsilylchloride and methanol providesthe methyl ester (18). Activation of the free hydroxyl to thechloroformate with phosgene in dichloromethane followed by addition ofmorpholine, then hydrolysis provides morpholine-4-carboxylicacid-1S-carboxy-2-cyclohexyl ethyl ester (19). Coupling of (19) to thesolid phase construct (9) followed by cleavage, provides (20), anexample of general formula (I) where R³=‘H’, (X)_(o)‘-’, (W)_(n)=‘O’,n=1, (V)_(m)=‘CO’ and U=morpholino. To those skilled in the practices oforganic synthesis, a wide variety of α-hydroxyacid esters such as (18)could be converted to the activated chloroformate following the generalconditions detailed. Additionally, morpholine may be replaced by anyreasonable amine, providing many variations of carboxylic acid (19)following the general conditions detailed. Thus analogues of (20)exploring a wide range of (V)_(m) and U in general formula (I) may beprepared through the general conditions detailed in Scheme 5.

[0188] Alternatively, (Scheme 6), a wide range of alkylsuccinate estersexemplified by 2R-cyclohexylmethylsuccinic acid 1-methyl ester (21) arecommercially available or readily prepared by known methods (see (a)Azam et al, J. Chem Soc. Perkin Trans. 1, 621-, 1996; (b) Evans et al,J. Chem. Soc. Perkin Trans. 1, 103, 2127, 1981; (c) Oikawa et al, Tet.Lett, 37, 6169, 1996). Carboxyl activation of alkylsuccinate ester (21)followed by addition of morpholine in dimethylformamide and subsequentester hydroylsis, provides2R-cyclohexylmethyl-4-morpholinyl-4-oxo-butyric acid (22). Coupling of(22) to the solid phase construct (9) followed by cleavage, provides(23), an example of general formula (I) where R³=‘H’, (X)_(o)=‘CH₂’ i.e.R⁹, R¹⁰=‘H’, o=1, (W)_(n)=‘CO’, n=1, (V)_(m)=‘-’ and U=morpholino. Tothose skilled in the practices of organic synthesis, a wide variety ofalkylsuccinate esters such as (21) may be prepared and converted to thecorresponding substituted alkylsuccinate acid such as (22) following thegeneral conditions detailed. Additionally, morpholine may be replaced byany reasonable amine, providing many variations of carboxylic acid (22)following the general conditions detailed. Thus analogues of (23)exploring a wide range of (X)_(o), (V)_(m) and U in general formula (I)may be prepared through the general conditions detailed in Scheme 6.

[0189] Alternatively, (Scheme 7), a wide range of biarylalkylaceticacids, exemplified by 2RS-biphenyl-3-yl-4-methylpentanoic acid (25) arereadily available by known methods (see (a) DesJarlais, R L. et al, J.Am. Chem. Soc, 120, 9114-9115, 1998; (b) Oballa, R. M. et al, WO0149288). Coupling of biarylalkylacetic acid (25) to the solid phaseconstruct (9) followed by cleavage, provides (26), an example of generalformula (I) where R³=‘H’, (X)_(o)=‘-’, (W)_(n)=‘-’, (V)_(m)=‘-’ andU=m-biphenyl. To those skilled in the practices of organic synthesis, awide variety of biarylalkylacetic acids such as (25) may be prepared byalkylation of the α-anion of the free acid analogue of (24), which inturn is prepared by Suzuki coupling of phenylboronic acid and3-bromophenylacetic acid methyl ester. Phenylboronic acid may bereplaced by a wide range of arylboronic acids in the Suzuki coupling,providing many variations of carboxylic acid (25) following the generalconditions detailed. Thus analogues of (26) exploring a wide range ofgroup ‘U’ in general formula (I) may be prepared through the generalconditions detailed in Scheme 7.

[0190] Many other possibilities for solid phase organic chemistry (e.g.see Brown, R. D. J. Chem. Soc., Perkin Trans. 1, 19, 3293-3320, 1998,for a review of recent SPOC publications) can be used to derivatiseconstruct (9) towards compounds of general formula (I). For example, theleft-hand portion ‘U-V-W-X-Y’ in general formula (I) can be partiallyconstructed in solution, coupled to construct (9) and further modifiedon the solid phase (Scheme 8). For instance, a simple extension ofScheme 4 is through the oxidation of the intermediate solid phase boundspecies, with m-chloroperbenzoic acid in dichloromethane prior tocleavage, to give the sulphone analogue (27). As described in Scheme 4,many variations of carboxylic acid (16) may be prepared following thegeneral conditions detailed. Thus analogues of (27) exploring a widerange of (V)_(m) and U in general formula (I) may be prepared throughthe general conditions detailed in Schemes 4 and 8.

[0191] Compounds of general formula (I) can be finally released from thesolid phase by treatment with trifluoroacetic acid/water, followed byevaporation, lyophylisation and standard analytical characterisation.

[0192] A second strategy for the synthesis of compounds of generalformula (I) comprises:—

[0193] (f) Preparation of an appropriately functionalised and protected(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide or a(2-alkyl-4-oxo-tetrahydrothiophen-3-yl)amide or a(2-alkyl-5-oxocyclopentyl)amide building block in solution. Preferredprotecting groups for solution phase chemistry are theNα-tert-butoxycarbonyl group and the Nα-benzyloxycarbonyl group.

[0194] (g) Standard organic chemistry methods for the conversion ofbuilding block (f) towards compounds of general formula (I).

[0195] In the simplest example, the entire left hand portion of aninhibitor of general formula (I) can be prepared in solution bytraditional organic chemistry methods and coupled to building block (f)(see Scheme 9 exemplified by preparation and use of the(2-alkyl-4-oxo-tetrahydrofuran-3-yl)carbamic acid tert-butyl ester(30)).

[0196] The general strategy detailed in Scheme 9 is particularly usefulwhen the compound of general formula (I) contains a substituent that islabile to trifluoroacetic acid, this being the final reagent used ineach of the solid phase Schemes 3-8. For example (Scheme 10), treatmentin solution of α-hydroxyacid (32) with sodium hydride in adimethylformamide/dichloromethane mixture followed by addition of4-tert-butylbenzyl bromide, provides2RS-(4-tert-butylbenzyloxy)-4-methylpentanoic acid (33). Coupling of(33) to hydrochloride salt (31), provides (34), an example of generalformula (I) where R³=‘H’, (X)_(o)=‘-’, (W)_(n)=‘O’, n=1, (V)_(m)=‘CH₂’,i.e. R¹², R¹³=‘H’, m=1 and U=4-tert-butylphenyl. To those skilled in thepractices of organic synthesis, 4-tert-butylbenzyl bromide may bereplaced by any reasonable Ar—CR¹²R¹³-halogen, providing many variationsof carboxylic acid (33) under the conditions shown. Thus analogues of(34) exploring a wide range of (V)_(m) and U in general formula (I) maybe prepared through the conditions detailed in Scheme 10.

[0197] A third strategy for the synthesis of compounds of generalformula (I) where the addition of U-V-W-X-Y to the protected(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide or a(2-alkyl-4-oxo-tetrahydrothiophen-3-yl)amide or a (2-alkyl-5-oxocyclopentyl)amide building block involves multistep organic reactionscomprises:—

[0198] (h) Preparation of an appropriately functionalised and protected(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide or a(2-alkyl-4-oxo-tetrahydrothiophen-3-yl)amide or a(2-alkyl-5-oxocyclopentyl)amide building block in solution. Preferredprotecting groups for solution phase chemistry are theNα-tert-butoxycarbonyl group and the Nα-benzyloxycarbonyl group.

[0199] (i) Protection of the ketone functionality of the(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amide or a(2-alkyl-4-oxo-tetrahydrothiophen-3-yl)amide or a(2-alkyl-5-oxocyclopentyl)amide building block e.g. as a dimethylacetal.Alternatively, the ketone may be reduced to the achiral secondaryalcohols and re-oxidised as the final synthetic step.

[0200] (j) Standard organic chemistry methods for the conversion ofbuilding block (i) towards compounds of general formula (I).

[0201] Intermediates may be prepared in solution, followed by couplingto building block (i) and further derivitisation towards compounds ofgeneral formula (I) (see Scheme 11 exemplified by preparation and use ofthe (4-Hydroxy-2-alkyl-tetrahydrofuran-3-yl) carbamic acid tert-butylester (35)).

[0202] Alternatively, depending upon the types of chemistry used toconstruct the left hand side U-V-W-X-Y of compounds of general formula(I), the ketone may require protection e.g. as the dimethyl acetal. Sucha method is detailed and exemplified in Scheme 12 by the preparation anduse of (4,4-Dimethoxy-2-alkyl-tetrahydrofuran-3-yl)-carbamic acid benzylester (38).

[0203] The invention extends to novel intermediates as described above,and to processes for preparing compounds of general formula (I) fromeach of its immediate precursors. In turn, processes for preparingintermediates from their immediate precursors also form part of theinvention.

[0204] Compounds of general formula (I) are useful both as laboratorytools and as therapeutic agents. In the laboratory certain compounds ofthe invention are useful in establishing whether a known or newlydiscovered cysteine protease contributes a critical or at leastsignificant biochemical function during the establishment or progressionof a disease state, a process commonly referred to as ‘targetvalidation’.

[0205] According to a second aspect of the invention, there is provideda method of validating a known or putative cysteine protease inhibitoras a therapeutic target, the method comprising:

[0206] (a) assessing the in vitro binding of a compound as describedabove to an isolated known or putative cysteine protease, providing ameasure of potency; and optionally, one or more of the steps of:

[0207] (b) assessing the binding of the compound to closely relatedhomologous proteases of the target and general house-keeping proteases(e.g. trypsin) to provides a measure of selectivity;

[0208] (c) monitoring a cell-based functional marker of a particularcysteine protease activity, in the presence of the compound; and

[0209] (d) monitoring an animal model-based functional marker of aparticular cysteine protease activity in the presence of the compound.

[0210] The invention therefore provides a method of validating a knownor putative cysteine protease inhibitor as a therapeutic target.Differing approaches and levels of complexity are appropriate to theeffective inhibition and ‘validation’ of a particular target. In thefirst instance, the method comprises assessing the in vitro binding of acompound of general formula (I) to an isolated known or putativecysteine protease, providing a measure of ‘potency’. An additionalassessment of the binding of a compound of general formula (I) toclosely related homologous proteases of the target and generalhouse-keeping proteases (e.g. trypsin) provides a measure of‘selectivity’. A second level of complexity may be assessed bymonitoring a cell-based functional marker of a particular cysteineprotease activity, in the presence of a compound of general formula (I).For example, a ‘human osteoclast resorption assay’ has been utilised asa cell-based secondary in vitro testing system for monitoring theactivity of cathepsin K and the biochemical effect of proteaseinhibitors (e.g. see WO-A-9850533). An ‘MHC-II processing—T-cellactivation assay’ has been utilised as a cell-based secondary in vitrotesting system for monitoring the activity of cathepsin S and thebiochemical effect of protease inhibitors (Shi, G-P., et al, Immunity,10, 197-206, 1999). When investigating viral or bacterial infectionssuch a marker could simply be a functional assessment of viral (e.g.count of mRNA copies) or bacterial loading and assessing the biochemicaleffect of protease inhibitors. A third level of complexity may beassessed by monitoring an animal model-based functional marker of aparticular cysteine protease activity, in the presence of a compound ofgeneral formula (I). For example, murine models of Leishmania infection,P. vinckei infection, malaria (inhibition of falcipain) and T. cruziinfection (cruzipain), indicate that inhibition of cysteine proteasesthat play a key role in pathogen propagation is effective in arrestingdisease symptoms, ‘validating’ said targets.

[0211] The invention therefore extends to the use of a compound ofgeneral formula (I) in the validation of a known or putative cysteineprotease inhibitor as a therapeutic target.

[0212] Compounds of general formula (I) are useful for the in vivotreatment or prevention of diseases in which participation of a cysteineprotease is implicated.

[0213] According to a third aspect of the invention, there is provided acompound of general formula (I) for use in medicine, especially forpreventing or treating diseases in which the disease pathology may bemodified by inhibiting a cysteine protease.

[0214] According to a fourth aspect of the invention, there is providedthe use of a compound of general formula (I) in the preparation of amedicament for preventing or treating diseases in which the diseasepathology may be modified by inhibiting a cysteine protease.

[0215] Certain cysteine proteases function in the normal physiologicalprocess of protein degradation in animals, including humans, e.g. in thedegradation of connective tissue. However, elevated levels of theseenzymes in the body can result in pathological conditions leading todisease. Thus, cysteine proteases have been implicated in variousdisease states, including but not limited to, infections by Pneumocystiscarinii, Trypsanoma cruzi, Trypsanoma brucei brucei and Crithidiafsiculata; as well as in osteoporosis, autoimmunity, schistosomiasis,malaria, tumour metastasis, metachromatic leukodystrophy, musculardystrophy, amytrophy, and the like. See WO-A-9404172 and EP-A-0603873and references cited in both of them. Additionally, a secreted bacterialcysteine protease from S. Aureus called staphylopain has been implicatedas a bacterial virulence factor (Potempa, J., et al. J. Biol. Chem,262(6), 2664-2667,1998).

[0216] The invention is useful in the prevention and/or treatment ofeach of the disease states mentioned or implied above. The presentinvention also is useful in a methods of treatment or prevention ofdiseases caused by pathological levels of cysteine proteases,particularly cysteine proteases of the papain superfamily, which methodscomprise administering to an animal, particularly a mammal, mostparticularly a human, in need thereof a compound of the presentinvention. The present invention particularly provides methods fortreating diseases in which cysteine proteases are implicated, includinginfections by Pneumocystis carinii, Trypsanoma cruzi, Trypsanoma brucei,Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus,foot-and-mouth disease virus and Crithidia fusiculata; as well as inosteoporosis, autoimmunity, schistosomiasis, malaria, tumour metastasis,metachromatic leukodystrophy, muscular dystrophy and amytrophy.

[0217] Inhibitors of cruzipain, particularly cruzipain-specificcompounds, are useful for the treatment of Chagas' disease.

[0218] In accordance with this invention, an effective amount of acompound of general formula (I) may be administered to inhibit theprotease implicated with a particular condition or disease. Of course,this dosage amount will further be modified according to the type ofadministration of the compound. For example, to achieve an “effectiveamount” for acute therapy, parenteral administration of a compound ofgeneral formula (I) is preferred. An intravenous infusion of thecompound in 5% dextrose in water or normal saline, or a similarformulation with suitable excipients, is most effective, although anintramuscular bolus injection is also useful. Typically, the parenteraldose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and20 mg/kg, in a manner to maintain the concentration of drug in theplasma at a concentration effective to inhibit a cysteine protease. Thecompounds may be administered one to four times daily at a level toachieve a total daily dose of about 0.4 to about 400 mg/kg/day. Theprecise amount of an inventive compound which is therapeuticallyeffective, and the route by which such compound is best administered, isreadily determined by one of ordinary skill in the art by comparing theblood level of the agent to the concentration required to have atherapeutic effect. Prodrugs of compounds of the present invention maybe prepared by any suitable method. For those compounds in which theprodrug moiety is a ketone functionality, specifically ketals and/orhemiacetals, the conversion may be effected in accordance withconventional methods.

[0219] The compounds of this invention may also be administered orallyto the patient, in a manner such that the concentration of drug issufficient to inhibit bone resorption or to achieve any othertherapeutic indication as disclosed herein. Typically, a pharmaceuticalcomposition containing the compound is administered at an oral dose ofbetween about 0.1 to about 50 mg/kg in a manner consistent with thecondition of the patient. Preferably the oral dose would be about 0.5 toabout 20 mg/kg.

[0220] No unacceptable toxicological effects are expected when compoundsof the present invention are administered in accordance with the presentinvention. The compounds of this invention, which may have goodbioavailability, may be tested in one of several biological assays todetermine the concentration of a compound which is required to have agiven pharmacological effect.

[0221] According to a fifth aspect of the invention, there is provided apharmaceutical or veterinary composition comprising one or morecompounds of general formula (I) and a pharmaceutically or veterinarilyacceptable carrier. Other active materials may also be present, as maybe considered appropriate or advisable for the disease or conditionbeing treated or prevented.

[0222] The carrier, or, if more than one be present, each of thecarriers, must be acceptable in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient.

[0223] The formulations include those suitable for rectal, nasal,topical (including buccal and sublingual), vaginal or parenteral(including subcutaneous, intramuscular, intravenous and intradermal)administration, but preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, e.g. tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

[0224] Such methods include the step of bringing into association theabove defined active agent with the carrier. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active agent with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product. Theinvention extends to methods for preparing a pharmaceutical compositioncomprising bringing a compound of general formula (I) in conjunction orassociation with a pharmaceutically or veterinarily acceptable carrieror vehicle.

[0225] Formulations for oral administration in the present invention maybe presented as: discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active agent; as a powderor granules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion; or as a bolus etc.

[0226] For compositions for oral administration (e.g. tablets andcapsules), the term “acceptable carrier” includes vehicles such ascommon excipients e.g. binding agents, for example syrup, acacia,gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone),methylcellulose, ethylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

[0227] A tablet may be made by compression or moulding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

[0228] Other formulations suitable for oral administration includelozenges comprising the active agent in a flavoured base, usuallysucrose and acacia or tragacanth; pastilles comprising the active agentin an inert base such as gelatin and glycerin, or sucrose and acacia;and mouthwashes comprising the active agent in a suitable liquidcarrier.

[0229] Parenteral formulations will generally be sterile.

[0230] According to a sixth aspect of the invention, there is provided aprocess for the preparation of a pharmaceutical or veterinarycomposition as described above, the process comprising bringing theactive compound(s) into association with the carrier, for example byadmixture.

[0231] Preferred features for each aspect of the invention are as foreach other aspect mutatis mutandis.

[0232] The invention will now be illustrated with the followingexamples:

[0233] Solution Phase Chemistry—General Methods

[0234] All solvents were purchased from ROMIL Ltd (Waterbeach,Cambridge, UK) at SpS or Hi-Dry grade unless otherwise stated. Generalpeptide synthesis reagents were obtained from Chem-Impex Intl. Inc.(Wood Dale Ill. 60191. USA). Thin layer chromatography (TLC) wasperformed on pre-coated plates (Merck aluminium sheets silica 60 F254,part no. 5554). Visualisation of compounds was achieved underultraviolet light (254 nm) or by using an appropriate staining reagent.Flash column purification was performed on silica gel 60 (Merck 9385).All analytical HPLC were obtained on Phenomenex Jupiter C₄, 5μ, 300 A,250×4.6 mm, using mixtures of solvent A=0.1% aq trifluoroacetic acid(TFA) and solvent B=90% acetonitrile/10% solvent A on automated Agilentsystems with 215 and/or 254 nm UV detection. Unless otherwise stated agradient of 10-90% B in A over 25 minutes at 1.5 mL/min was performedfor full analytical HPLC analysis. HPLC-MS analysis was performed on anAgilent 1100 series LC/MSD, using automated Agilent HPLC systems, with agradient of 10-90% B in A over 10 minutes on Phenomenex Columbus C₈, 5μ, 300 A, 50×2.0 mm at 0.4 mL/min. Nuclear magnetic resonance (NMR) wereobtained on a Bruker DPX400 (400 MHz 1H frequency; QXI probe) in thesolvents and temperature indicated. Chemical shifts are expressed inparts per million (δ) and are referenced to residual signals of thesolvent. Coupling constants (J) are expressed in Hz.

[0235] Example inhibitors (1-47) were prepared through a combination ofsolution chemistry and solid phase Fmoc-based chemistries (see ‘SolidPhase Peptide Synthesis’, Atherton, E. and Sheppard, R. C., IRL PressLtd, Oxford, UK, 1989, for a general description) (Schemes 1-12).

[0236] Solid Phase Chemistry—General Methods

[0237] An appropriately protected and functionalised building block wasprepared in solution (e.g. general compound (6), Scheme 1), thenreversibly attached to the solid phase through an appropriate linker.Rounds of coupling/deprotection/chemical modification e.g. oxidation,were then performed until the full length desired molecule was complete(Scheme 2). Example inhibitors (1-47) were then released (cleaved) fromthe solid phase, analysed, purified and assayed for inhibition verses arange of proteases.

[0238] Generally, multipins (polyamide 1.2→10 μmole loadings, seewww.mimotopes.com) were used for the solid phase synthesis, although anysuitable solid phase surface could be chosen. In general, the 1.2 μmolegears were used to provide small scale crude examples for preliminaryscreening, whilst the 10 μmole crowns were used for scale-up synthesisand purification of preferred examples. Standard coupling and Fmocdeprotection methods were employed (see Grabowska, U. et al, J. Comb.Chem. 2(5), 475-490, 2000. for a thorough description of solid phasemultipin methodologies).

[0239] Preparation of Initial Assembly

[0240] Building Block-linker constructs (e.g. (8), typically 100 mg to 2g) were carboxyl activated with2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 1 mole equivalent), 1-hydroxybenzotriazole.hydrate(HOBT, 1 mole equivalent) and N-methylmorpholine (NMM, 2 moleequivalents) in dimethylformamide (DMF, typically 1 to 10 mL) for 5minutes. Amino functionalised DA/MDA crowns or HEMA gears (10 μmole percrown/1.2 μmole per gear, 0.33 mole equivalent of total surface aminofunctionalisation compared to activated construct) were added, followedby additional DMF to cover the solid phase surface. The loading reactionwas left overnight. Following overnight loading, crowns/gears were takenthrough standard cycles washing, Fmoc deprotection and loadingquantification (see Grabowska, U. et al) to provide loaded BuildingBlock-linker constructs (e.g. (9)). Analysis indicated virtuallyquantitative loading in all examples.

[0241] Coupling Cycles

[0242] The coupling of standard Fmoc-aminoacids and novel carboxylicacids (e.g. (12) Scheme 3, (16) Scheme 4, (19) Scheme 5, (22) Scheme 6,(25) Scheme 7) (10 or 20 mole equivalent) were performed via carboxylactivated with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU, 10 or 20 mole equivalent),1-hydroxybenzotriazole.hydrate (HOBT, 10 or 20 mole equivalent) andN-methylmorpholine (NMM, 20 or 40 mole equivalents) indimethylformamide, with pre-activation for 5 minutes. Activated specieswere dispensed to the appropriate wells of a polypropylene 96-well plate(Beckman, 1 mL wells, 500 μL solution per well for crowns or 250 μLsolution per well for gears) in a pattern required for synthesis. Loadedfree amino Building Block-linker constructs (e.g. (9)) were added andthe coupling reaction left overnight. Following overnight coupling,crowns/gears were taken through standard cycles washing and Fmocdeprotection (see Grabowska, U. et al.). Identical activation andcoupling conditions were used for the coupling of a range of carboxylicacids (R—COOH). Alternatively, chloroformates e.g.morpholine-4-carbonylchloride (10 mole equivalent), were coupled in DMFwith the addition of NMM (10 mole equivalents).

[0243] Acidolytic Cleavage Cycle

[0244] A mixture of 95% TFA/5% water was pre-dispensed into twopolystyrene 96-well plates (Beckman, 1 mL wells, 600 μL solution perwell for crowns or 300 μL solution per well for gears) in a patterncorresponding to that of the synthesis. The completed multipin assemblywas added to the first plate (mother plate), the block covered in tinfoil and cleaved for 2 hours. The cleaved multipin assembly was thenremoved from the first plate and added to the second plate (washingplate) for 15 minutes. The spent multipin assembly was then discardedand the mother/washing plates evaporated on a HT-4 GeneVac plateevaporator.

[0245] Analysis and Purification of Cleaved Examples

[0246] (a) Ex 1.2 μmole Gears. 100 μL dimethylsulphoxide (DMSO) wasadded to each post cleaved and dried washing plate well, thoroughlymixed, transferred to the corresponding post cleaved and dried motherplate well and again thoroughly mixed. 10 μL of this DMSO solution wasdiluted to 100 μL with a 90% acetonitrile/10% 0.1% aq TFA mixture. 20 μLaliquots were analysed by HPLC-MS and full analytical HPLC. In each casethe crude example molecules gave the expected [M+H]⁺ ion and an HPLCpeak at >80% (by 215 nm UV analysis). This provided an approximately 10mM DMSO stock solution of good quality crude examples for preliminaryprotease inhibitory screening.

[0247] (b) Ex 10 mole Crowns. 500 μL of a 90% acetonitrile/10% 0.1% aqTFA mixture was added to each washing plate well, thoroughly mixed,transferred to the corresponding mother plate well and again thoroughlymixed. 5 mL of this solution was diluted to 100 μL with a 90%acetonitrile/10% 0.1% aq TFA mixture. 20 μL aliquots were analysed byHPLC-MS and full analytical HPLC. In each case the crude examplemolecules gave the expected [M+H]⁺ ion and an HPLC peak at >80% (by 215nm UV analysis). The polystyrene blocks containing crude examples werethen lyophilised.

[0248] (c) Individual examples (ex (b)) were re-dissolved in a 1:1mixture of 0.1% aq TFA/acetonitrile (1 mL) and purified bysemi-preparative HPLC (Phenomenex Jupiter C₄, 5μ, 300 A, 250×10 mm, a25-90% B in A gradient over 25 mins, 4.0 mL/min, 215 nm UV detection).Fractions were lyophilised into pre-tarred glass sample vials to providepurified examples (typically 2 to 4 mg, 40 to 80% yield).

[0249] (d) Purified examples were dissolved in an appropriate volume ofDMSO to provide a 10 mM stock solution, for accurate protease inhibitoryscreening.

EXAMPLE 1 (2S, 3S) 2-Benzyloxy-3-cyclohexyl-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0250]

[0251] Following the general details from Scheme 1, the required bicyclebuilding block (2S, 3S) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamicacid 9H-fluoren-9-ylmethyl ester (6a) was prepared as follows:

[0252] (1) Preparation of (1S, 1′S)[3-diazo-1-(1-tert-butoxyethyl)-2-oxo-propyl]-carbamic acid9H-fluoren-9-ylmethyl ester.

[0253] A solution of iso-butyl chloroformate (1.05 g, 7.7 mmol) indichloromethane (14 ml) and a solution of 4-methylmorpholine (1.42 g, 14mmol) in dichloromethane (14 ml) were simultaneously added to a stirredsuspension of Fmoc-O-(tert-butyl ether)-L-allo-threonine (2.78 g, 7mmol) in dichloromethane (70 ml) at −15° C. over 10 minutes under anatmosphere of nitrogen. Ethereal diazomethane [generated from diazald(7.14 g, ˜21 mmol) addition in diethyl ether (115 ml) to sodiumhydroxide (8.022 g) in water (11 ml)/ethanol (31 ml) at 60° C.] was thencautiously added and the resulting yellow solution was stirred at roomtemperature for 90 minutes. Acetic acid (˜3 ml) was cautiously added(until effervescence had ceased) then the mixture was diluted withtert-butyl methyl ether (70 ml). The ethereal layer was washed withwater (3×70 ml), dried (Na₂SO₄) and the solvent removed in vacuo toleave (1S, 1′S) [3-diazo-1-(1-tert-butoxyethyl)-2-oxo-propyl]-carbamicacid 9H-fluoren-9-ylmethyl ester as a yellow oil (5.47 g) which was usedwithout further purification.

[0254] (2) Cyclisation to (2S, 3S)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic acid 9H-fluoren-9-ylmethylester.

[0255] A solution of lithium chloride (2.97 g, 70 mmol) in 80% aceticacid in water (105 ml) was added to (1S, 1′S)[3-diazo-1-(1-tert-butoxyethyl)-2-oxo-propyl]carbamic acid9H-fluoren-9-ylmethyl ester (5.7 g, ˜7 mmol). The yellow oily suspensionwas stirred for 2 hours whereupon a pale yellow solution formedaccompanied by the evolution of a gas. The solvents were removed invacuo then the residue was dissolved in ethyl acetate (42 ml), washedwith 10% aqueous sodium carbonate solution (2×42 ml) and saturatedaqueous sodium chloride solution (42 ml). The combined aqueous layerswere extracted with ethyl acetate (2×42 ml) then the combined ethylacetate layers were dried (Na₂SO₄) and the solvents removed in vacuo toleave a yellow gum (4.28 g). The yellow gum was purified bychromatography over silica gel eluting with a gradient ofn-heptane:ethyl acetate 9:1→7:3. Appropriate fractions were combined andthe solvents removed in vacuo to leave (2S, 3S)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic acid 9H-fluoren-9-ylmethylester as a white crystalline solid (1.58 g, 67% from starting acid). TLC(single UV spot, Rf=0.27, n-heptane:ethyl acetate 2:1), analytical HPLCpeak Rt=17.92 mins, HPLC-MS (single UV peak with Rt=8.47 mins, 360.0[M+Na]⁺, 361.0 [M+H+Na]⁺).

[0256]_(δ)H (CDCl₃ at 298K); 1.28-1.50 (3H, CH ₃CHO, brs), 3.60-3.75(1H, CH₃CHO m), 3.78-4.00 (2H, Fmoc CH ₂, m), 4.04-4.24 (2H, Fmoc H-9and NHCHCO, m), 4.26-4.50 (2H, OCH ₂CO, dd), 4.92-5.14 (1H, NH, brs),7.17-7.36 (4H, ArH, Fmoc H-2, H-3, H-6 and H-7), 7.40-7.56 (2H, ArH,Fmoc H-1 and H-8), 7.60-7.75 (2H, ArH, Fmoc H-4 and H-5).

[0257]_(δ)C (CDCl₃ at 298K); 19.44 (u, CH₃CHO), 47.52 (u, Fmoc C-9),63.09 (u, NHCHCO), 67.65 (d, Fmoc CH₂), 71.20 (d, COCH₂), 77.82 (u, CH₃CHO), 120.43 (u, Fmoc C-4 and C-5), 125.38 (u, Fmoc C-1 and C-8), 127.51(u, Fmoc C-2 and C-7), 128.19 (u, Fmoc C-3 and C-6), 141.75 (q, Fmoc C4′and C-5′), 143.98/144.03 (q, Fmoc C-1′ and C-8′), 156.42 (q, OCON),212.18 (q, COCH₂).

[0258] Following the general details from Scheme 2, the required bicyclebuilding (2S, 35) (2-methyl-4oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6a) was converted to building block-linkerconstruct (8a) as follows:

[0259] (2S, 3S) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6a) (354 mg, 1.05 mmol, 1 eq) was dissolvedin a mixture of ethanol (9.6 mL) and water (1.4 mL) containing sodiumacetate.trihydrate (218 mg, 1.6 mmol, 1.5 eq).4-[[(hydrazinocarbonyl)amino] methyl]cyclohexanecarboxylic acid.trifluoroacetate (346 mg, 1.05 mmol, 1 eq, Murphy, A. M. et al., J. Am.Chem. Soc., 114, 3156-3157, 1992) was added and the mixture refluxed for2 hr. Chloroform (100 mL) was added and the organics washed with diluteaqueous hydrochloric acid (0.1 M, 1×100 mL). The acidic layer wasbackwashed with chloroform (3×100 mL) and the combined organics washedwith brine (100 mL), dried (Na₂SO₄) and evaporated under reducedpressure to afford linker construct (8a) as a white solid (630 mg).Analytical HPLC indicated one main peak at R_(t)=17.17 min, HPLC-MS(main UV peak with R_(t)=7.91 min, 535.3 [M+H]⁺. Crude (8a) was useddirectly for construct loading.

[0260] Following the general details from Scheme 2, the requiredbuilding block-linker construct (8a) was attached to the solid phaseproviding loaded building block-linker construct (9a) as follows:

[0261] Building block-linker construct (8a) (1.0 mmole),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro phosphate(HBTU, 379 mg, 1.0 mmole), 1-hydroxybenzotriazole.hydrate and (HOBT, 153mg, 1.0 mmole) were dissolved in dimethylformamide (5 mL) andN-methylmorpholine (NMM, 220 μL, 2.0 mmole) added. After pre-activationfor 5 minutes, free amine gears (250×1.2 μmole) were added, followed bydimethylformamide (30 mL) and left overnight. The spent couplingsolution was then added to free amine crowns (25×10 μmole) and leftovernight. Standard washing and analyses indicated loading at >95%.

[0262] The required ether carboxylic acid building block2RS-Benzyloxy-3-cyclohexylpropionic acid (compound (12), Scheme 3) wasprepared as follows:

[0263] (1) Preparation of 3-Cyclohexyl-2S-hydroxypropionic acid(Compound (11) Scheme 3)

[0264] A solution of sodium nitrite (12.1 g, 175 mmol) in water (40 ml)was added dropwise to a stirred suspension of(S)-α-aminocyclohexanepropionic acid hydrate (5 g, 26.5 mmol) in 0.5Msulphuric acid (120 ml, 60 mmol) at 0° C. over 1.5 hours. The mixturewas allowed to warm to ambient temperature over 20 hours. The productwas extracted into diethyl ether (2×25 ml) then the ethereal layers werewashed with saturated aqueous sodium chloride solution (2×25 ml), dried(Na₂SO₄) and the solvents removed in vacuo. The residue (5.3 g) wasrecrystallized from diethyl ether (10 ml) and heptane (25 ml) to give3-cyclohexyl-2S-hydroxypropionic acid as a white solid, yield 2.4 g,(53%).

[0265]_(δ)H (400 MHz, CDCl₃ at 298K), 0.89-1.35 (5H, m) and 1.51-1.86(7H, m) (OCHCH ₂ and cyclohexyl), 4.32 (1H, OCHCH₂, m)

[0266] (2) Preparation of 2RS-Benzyloxy-3-cyclohexylpropionic acid(Compound (12) Scheme 3)

[0267] Sodium hydride (265 mg of 60% dispersion in oil, 6.6 mmol) wasadded in two portions to a stirred mixture of3-cyclohexyl-2S-hydroxypropionic acid (0.52 g, 3.0 mmol),dimethylformamide (5 ml) and dichloromethane (5 ml) at 0° C. over 5minutes. The mixture was stirred at 0° C. for 5 minutes then at ambienttemperature for 45 minutes. Benzyl bromide (0.45 ml, 3.8 mmol) was addedthen the mixture stirred for 1 hour before adding dimethylformamide (5ml). After stirring for 4 hours potassium iodide (50 mg, 0.3 mmol) wasadded. The mixture was stirred for 20 hours then heated at 55° C. for 1hour then allowed to cool to ambient temperature and poured into water(15 ml). A saturated aqueous sodium chloride solution (5 ml) was addedthen the mixture was extracted with dichloromethane (5 ml then 10 ml)that was discarded. The aqueous layer was acidified using 1Mhydrochloric acid (10 ml) then extracted with dichloromethane (2×10 ml).The dichloromethane layer was dried (MgSO₄) and the solvent removed invacuo. The residue (0.55 g) was dissolved in dimethylformamide (8 ml)then cooled to 0° C. before adding sodium hydride (190 mg of 60%dispersion in oil, 4.75 mmol). The mixture was stirred for 30 minutesthen polymer bound isocyanate (380 mg, 2 mmolNg⁻¹) added. The mixturewas stirred for 2 hours at ambient temperature then poured into water(15 ml). 1M Hydrochloric acid (10 ml) was added then the product wasextracted into dichloromethane (2×10 ml), dried (Na₂SO₄) and the solventremoved in vacuo. The residue was purified by flash chromatography oversilica gel eluting with a gradient of methanol:dichloromethane 0:1→1:20.Appropriate fractions were combined and the solvents removed in vacuo togive 2RS-benzyloxy-3-cyclohexylpropionic acid as a colourless oil, yield41 mg (5.2%). HPLC-MS (single main UV peak with Rt=9.47 mins, 261.2[M−H]⁻, 285.2[M+Na]⁺, 547.3[2M+Na]⁺).

[0268]_(δ)H (400 MHz, CDCl₃ at 298K), 0.72-1.03 (2H, cyclohexane, m),1.08-1.38 (3H, cyclohexane, m), 1.45-1.93 (6H+2Hβ, cyclohexane, m),3.93-4.18 (1Ha, OCHCO), 4.35-4.53 (1H, CH ₂O, d, J=11.52 Hz), 4.68-4.88(1H, CH ₂O, d, J=11.54 Hz), 7.20-7.47 (5H, ArH, m), 9.36 (1H, OH, brs).

[0269] Compound (12) was coupled under standard conditions to loadedbuilding block-linker construct (9a) (following standard removal ofFmoc), then cleaved to provide EXAMPLE 1. The crude example was analysed(see general techniques). HPLC Rt=20.74 mins (>90%), HPLC-MS 360.2[M+H]⁺, 741.4 [2M+Na]⁺.

[0270] The following examples (2-10) were prepared as detailed forEXAMPLE 1, coupling with the required carboxylic acid building blocks toprovide the full length molecule.

EXAMPLE 2 (2S, 3S)2-Benzyloxy-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-3-phenyl-propionamide

[0271]

[0272] HPLC Rt=18.34 mins (>90%), HPLC-MS 354.2 [M+H]⁺, 729.3 [2M+Na]⁺.

[0273] The required carboxylic acid, 2RS-benzyloxy-3-phenylpropionicacid was prepared following the general method described for compound(12) as a colourless oil, yield 62 mg. HPLC-MS (single main UV peak withRt=8.27 mins, 257.1 [M+H]⁺).

[0274]_(δ)H (CDCl₃ at 298K); 3.05-3.30 (2H, PhCH ₂, m), 4.15-4.30 (1H,OCHCO, m), 4.36-4.50 (1H, CH ₂O, d, J=11.83 Hz), 4.66-4.83 (1H, CH ₂°,d, J=11.85 Hz), 7.13-7.45 (10H, ArH, m), 10.07 (1H, OH, brs).

EXAMPLE 3 (2S, 3S)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0275]

[0276] HPLC Rt=19.74 mins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0277] The required ether carboxylic acid building block3-Cyclohexyl-2R-(furan-2-ylmethylsulfanyl)-propionic acid (compound(16), Scheme 4) was prepared as follows:

[0278] (1) Preparation of (S)-α-bromocyclohexanepropionic Acid

[0279] A solution of sodium nitrite (2.26 g, 32.7 mmol) in water (6 ml)was added drop-wise at 0° C. over 4 hours to a stirred mixture of(s)-α-aminocyclohexanepropionic acid (5 g, 29.2 mmol), potassium bromide(11.40 g, 95.8 mmol) and concentrated sulphuric acid (3.3 ml) in water(35 ml). The mixture was stirred for 5 hours at 0° C. then at ambienttemperature for 16 hours. The product was extracted into diethyl ether(4×50 ml) then the combined ethereal layers were washed with saturatedaqueous sodium chloride solution (2×50 ml), dried (MgSO₄) and thesolvent removed in vacuo. The residue was purified by chromatographyover silica gel eluting with a gradient of methanol:dichloromethane1:50→1:20. Appropriate fractions were combined and the solvents removedin vacuo to leave (S)-α-bromocyclohexanepropionic acid as a pale yellowoil (3.64 g, 53%). TLC (single spot, Rf=0.45, 10% methanol indichloromethane), HPLC-MS (single main peak with Rt=8.85 mins,234.1/236.1 [M+H])⁺).

[0280]_(δ)H (CDCl₃ at 298K); 0.83-1.88 (11H, CH(cyclohexane), CH₂(cyclohexane), m), 1.89-2.05 (2H, BrCHCH ₂, m), 4.32-4.44 (1H, BrCHCH₂(cyclohexane), m).

[0281]_(δ)C (CDCl₃ at 298K); 26.19126.29/26.48/26.58/26.79, 32.51 and34.28 (cyclohexane CH₂), 33.32 (CHCH₂CHCO), 42.40 (BrCHCH₂), 68.83(BrCH), 176.01 (CO).

[0282] (2) Preparation of3-Cyclohexyl-2R-(furan-2-ylmethylsulfanyl)-propionic Acid (Compound(16), Scheme 4)

[0283] (S)-α-bromocyclohexanepropionic acid (0.41 g, 1.75 mmol) andfuran-2-ylmethylthiol (0.2 g, 1.75 mmol) were dissolved indimethylformamide (10 ml) and purged with nitrogen for 10 minutes. Thesolution was cooled to 0° C. then triethylamine (0.244 ml, 1.75 mmol)was added drop-wise over 1 minute. The mixture was stirred at 0° C. for30 minutes then at ambient temperature for 16 hours. The solvent wasremoved in vacuo, and the residue purified by chromatography over gelsilica using methanol:dichloromethane 1:100 as eluent. Appropriatefractions were combined and the solvents removed in vacuo to leave3-Cyclohexyl-2R-(furan-2-ylmethylsulfanyl)-propionic acid (16) as alight brown oil (86 mg, 18%). Analytical HPLC peak Rt=18.68 mins. TLC(single spot, Rf=0.45, 10% methanol in dichloromethane), HPLC-MS (singlemain peak with Rt=9.43 mins, 291.1 [M+Na]⁺).

[0284]_(δ)H (CDCl₃ at 298K); 0.67-0.88 (2H, CH ₂(cyclohexane), m),0.98-1.76 (11H, CH(cyclohexane), CH ₂(cyclohexane), m), 3.20-3.32 (1H,SCHCO, t J=7.8 Hz), 3.69-3.79 (1H, CH ₂S, d, J=14.7 Hz), 3.85-3.95 (1H,CH ₂S, d, J=14.7 Hz), 6.14/6.28 (2H, furan H-3 and H-4, d), 7.30 (1H,furan H-5, s).

[0285]_(δ)C (CDCl₃ at 298K); 26.26/26.45/26.58126.77/26.89, 28.66, 33.11and 33.34 (each d, cyclohexane CH₂ and SCHCH₂), 35.52 (u, CHCH₂CHS),38.49 (d, SCH₂), 43.84 (u, SCH), 108.64 (u, furan C-3), 110.78 (u, furanC-4), 142.65 (u, furan C-5), 150.02 (q, furan C-2), 177.63 (q, CO).

EXAMPLE 4 (2S, 3S)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0286]

[0287] HPLC Rt=19.74 mins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0288] The required ether carboxylic acid building block3-Cyclohexyl-2S-(furan-2-ylmethylsulfanyl)-propionic acid (invertedisomer of compound (16), Scheme 4) was prepared as follows:

[0289] (1) Preparation of (R)-α-bromocyclohexanepropionic Acid

[0290] A solution of sodium nitrite (0.45 g, 6.5 mmol) in water (1.2 ml)was added dropwise at 0° C. over 4 hours to a stirred mixture of(R)-α-aminocyclohexanepropionic acid (1 g, 5.8 mmol), potassium bromide(2.3 g, 19.2 mmol) and concentrated sulphuric acid (0.66 ml) in water (7ml). The mixture was stirred for 5 hours at 0° C. then at ambienttemperature for 16 hours. The product was extracted into diethyl ether(4×20 ml) then the combined ethereal layers were washed with saturatedaqueous sodium chloride solution (2×50 ml), dried (MgSO₄) and thesolvent removed in vacuo. The residue was purified by chromatographyover silica gel eluting with a gradient of methanol:dichloromethane1:50→1:20. Appropriate fractions were combined and the solvents removedin vacuo to give (R)-α-bromocyclohexanepropionic acid as a pale yellowoil 0.374 g, (27%). TLC (single spot, Rf=0.45, 10% methanol indichloromethane), HPLC-MS (single main peak with Rt=8.88 mins,234.1/236.1 [M+H]⁺, 257.2/259.2 [M+Na]⁺).

[0291]_(δ)H (400 MHz, CDCl₃ at 298K), 0.83-1.88 (11H, CH(cyclohexane),CH ₂ (cyclohexane), m), 1.89-2.05 (2Hβ, m), 4.32-4.44 (1Hα, m).

[0292] (2) Preparation of3-Cyclohexyl-2S-(furan-2-ylmethylsulfanyl)-propionic Acid (InvertedIsomer of Compound (16), Scheme 4)

[0293] (R)-α-bromocyclohexanepropionic (0.37 g, 1.58 mmol) andfuran-2-yl methanethiol (0.18 g, 1.58 mmol) were dissolved indimethylformamide (10 ml) and purged with nitrogen for 10 minutes. Thesolution was cooled to 0° C. then triethylamine (0.22 ml, 1.58 mmol) wasadded drop-wise over 1 minute. The mixture was stirred at 0° C. for 30minutes then at ambient temperature for 16 hours. The solvent wasremoved in vacuo, and the residue purified by chromatography over gelsilica using methanol dichloromethane 1:100 as eluent. Appropriatefractions were combined and the solvents removed in vacuo to give3-Cyclohexyl-2S-(furan-2-ylmethylsulfanyl)-propionic acid as a lightbrown oil, yield 142 mg, (33%). Analytical HPLC peak Rt=18.68 mins. TLC(single spot, Rf=0.45, 10% methanol in dichloromethane), HPLC-MS (singlemain peak with Rt=9.53 mins, 291.0 [M+Na]⁺).

[0294]_(δ)H (400, CDCl₃ at 298K), 0.68-0.89 (2H, CH ₂(cyclohexane), m),0.97-1.77 (11H, CH(cyclohexane), C{overscore (H)}₂(cyclohexane), m),3.21-3.32 (1Hα, t, J=7.8 Hz), 3.69-3.79 (1H, CH ₂S, d, J=14.8 Hz),3.84-3.94 (1H, CH ₁S, d, J=14.8 Hz), 6.15/6.28 (2H, furan H-3 and H-4),7.30 (1H, furan H-5, s).

EXAMPLE 5 (2S, 3S)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0295]

[0296] HPLC Rt=17.60 mins (>80%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0297] The intermediate loaded thioether (1.2 μmole gear) of EXAMPLE 3was oxidised (Scheme 8) with m-chloroperbenzoic acid (5 eq, 65% reagent,1.6 mg) in dichloromethane (200 μL) for 5 hrs, followed by standardwashing and then cleaved to provide EXAMPLE 5.

EXAMPLE 6 (2S, 3S)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0298]

[0299] HPLC Rt=17.62 mins (>95%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0300] The intermediate loaded thioether (1.2 μmole gear) of EXAMPLE 4was oxidised (Scheme 8) with m-chloroperbenzoic acid (5eq, 65% reagent,1.6 mg) in dichloromethane (200 μL) for 5 hrs, followed by standardwashing and then cleaved to provide EXAMPLE 6.

EXAMPLE 7 (2S, 3S)2-(4-tert-Butyl-phenylmethanesulfonyl)-4-methyl-pentanoic acid(2-methyl-4oxo-tetrahydrofuran-3-yl)-amide

[0301]

[0302] HPLC Rt=21.14 mins (>95%), HPLC-MS 424.2 [M+H]⁺, 869.4 [2M+Na]⁺.

[0303] The required carboxylic acid building block2R-(4-tert-Butyl-benzylsulfanyl)-4-methyl-pentanoic acid (analogue ofcompound (16), Scheme 4) was prepared from (S)-2-bromo-4-methylpentanoicacid as follows:

[0304] (1) Preparation of (S)-2-bromo-4-methylpentanoic Acid

[0305] A solution of sodium nitrite (5.1 g, 73 mmol) in water (15 ml)was added drop-wise at 0° C. over 5 hours to a stirred mixture ofL-leucine (8.75 g, 67 mmol), potassium bromide (29.75 g, 0.25 mol) andconcentrated sulphuric acid (8.6 ml) in water (100 ml). The mixture wasstirred for 30 minutes at 0° C. then at ambient temperature for 20hours. The product was extracted into diethyl ether (2×150 ml) then thecombined ethereal layers were washed with saturated aqueous sodiumchloride solution (2×100 ml), dried (MgSO₄) and the solvent removed invacuo. The residue was purified by flash chromatography over silica geleluting with a gradient of methanol:dichloromethane 1:50→1:20.Appropriate fractions were combined and the solvents removed in vacuo toleave (S)-2-bromomethylpentanoic acid as a colourless oil (2.22 g, 17%).TLC (single spot, Rf=0.2, methanol:dichloromethane 1:20).

[0306]_(δ)H (CDCl₃ at 298K); 0.95 and 0.99 (both 3H, CH ₃CH, d, J=6.55Hz), 1.77-1.89 (1H, CH₃CH, m), 1.93 (2H, CH ₂H, m), 4.31 (1H, CHBr, t,J=7.7 Hz), 9.3 (1H, CO₂ H, brs).

[0307] Additional (S)-2-bromo-4-methylpentanoic acid was obtained (6.55g), however other components were observed by TLC analysis(methanol:dichloromethane 1:20).

[0308] (2) Preparation of2R-(4-tert-butylbenzylsulfanyl)-4-methylpentanoic Acid

[0309] A solution of (S)-2-bromo-4-methylpentanoic acid (1.1 g, 5.6mmol) and [4-(tert-butyl)phenyl]methanethiol (1.0 g, 5.6 mmol) indimethylformamide (15 ml) was purged with nitrogen for 5 minutes thencooled to 0° C. Triethylamine (0.79 ml, 5.7 mmol) was added drop-wiseover 1 minute then the mixture was stirred for two days at ambienttemperature. The solvents were removed in vacuo and residue purified byflash chromatography over silica gel eluting with a gradient ofmethanol:dichloromethane 0:1→1:20. Appropriate fractions were combinedand the solvents removed in vacuo to leave a residue which was purifiedby flash chromatography over silica gel eluting with ethylacetate:heptane 2:5. Appropriate fractions were combined and thesolvents removed in vacuo to leave2R-(4-tert-butylbenzylsulfanyl)-4-methylpentanoic acid as a colourlessoil (200 mg, 12%). TLC (single spot, Rf=0.2, heptane: ethyl acetate5:2), analytical HPLC with main peak Rt=22.117 mins, HPLC-MS (main UVpeak with Rt=11.072 mins, 317.2 [M+Na]⁺).

[0310]_(δ)H (CDCl₃ at 298K); 0.70 and 0.85 (both 3H, CH ₃CH, d, J=6.3),1.29 (9H, (CH ₃)₃C, s), 1.44-1.51 (1H, CH₃CH, m), 1.62-1.75 (2H, CH ₂CH,m), 3.15-3.20 (1H, SCH, m), 3.81 and 3.88 (both 1H, SCH ₂, d, J=13.2Hz), 7.25-7.35 (4H, aromatic).

[0311] The intermediate loaded thioether (1.2 μmole gear) was oxidised(Scheme 8) with m-chloroperbenzoic acid (5 eq, 65% reagent, 1.6 mg) indichloromethane (200 μL) for 5 hrs, followed by standard washing andthen cleaved to provide EXAMPLE 7.

EXAMPLE 8 (2S, 3S) Morpholine-4-carboxylic acid2-cyclohexyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylEster

[0312]

[0313] HPLC_MS 383.4 [M+H]⁺.

[0314] The required carboxylic acid building blockMorpholine-4carboxylic acid 1-carboxy-2-cyclohexyl-ethyl ester (compound(19), Scheme 5) was prepared as follows:

[0315] (1) Preparation of 3-cyclohexyl-2S-hydroxypropionic Acid MethylEster. (Compound (18), Scheme 5)

[0316] Trimethylsilyl chloride (1.78 ml, 14.0 mmol) was added dropwiseto a stirred solution of 3-cyclohexyl-2S-hydroxypropionic acid (compound(11) 1.31 g, 7.6 mmol) in methanol (35 ml). The mixture was stirred atroom temperature for 20 hours then the solvents were removed in vacuo toleave a pale yellow oil (1.38 g). The crude oil was purified bychromatography over silica gel eluting with a gradient of n-heptane:ethyl acetate (4:1) Appropriate fractions were combined and the solventsremoved in vacuo to give 3-cyclohexyl-2S-hydroxypropionic acid methylester as a colourless oil, yield 1.26 g, (89%). HPLC-MS (single mainpeak with Rt=7.43 mins, 187.14 [M+H]⁺, 395.2 [2M+Na]⁺).

[0317]_(δ)H (400 MHz, CDCl₃ at 298K), 0.84-1.04 (2H, CH ₂(cyclohexane),m), 1.09-1.36 (3H, CH ₂(cyclohexane), CH(cyclohexane), m), 1.45-1.77(7H, CH ₂(cyclohexane), CH(cyclohexane), m), 1.80-1.90 (1Hβ, m), 2.72(1H, OH, d, J=5.99 Hz), 3.80 (3H, CH ₃O, s), 4.20-4.31 (1Hα, m).

[0318] (2) Preparation of Morpholine-4-carboxylic Acid1-carboxy-2-cyclohexylethyl Ester (Compound (18), Scheme 5)

[0319] A solution of phosgene (33.6 ml, 20% in toluene) was added to3-cyclohexyl-2S-hydroxypropionic acid methyl ester (compound (11), 0.7g, 4.03 mmol) followed by 6 drops of dimethylformamide. The mixture wasstirred at ambient temperature for 16 hours then the solvents removed invacuo. The residue was azeotroped with toluene (3×20 ml), and dissolvedin anhydrous dichloromethane (11 ml). The solution was cooled to 0° C.then morpholine (0.86 g, 9.85 mmol) added. The mixture was stirred for 2hours then partitioned between dichloromethane (30 ml) and 0.5Mhydrochloric acid (30 ml). The dichloromethane layer was washed withsaturated aqueous sodium hydrogen carbonate solution (30 ml), saturatedaqueous sodium chloride solution (30 ml), dried (Na₂SO₄) and the solventremoved in vacuo. The residue was purified by chromatography over silicagel eluting with ethyl acetate: heptane 1:1. Appropriate fractions werecombined and the solvents removed in vacuo to leavemorpholine-4-carboxylic acid 2S-cyclohexyl-1-methoxycarbonylethyl ester(0.13 g, 10%) as an oil. A solution of lithium hydroxide monohydrate(17.5 mg, 0.418 mmol) in water (0.76 ml) was added to an iced-waterchilled solution of morpholine-4-carboxylic acid2-cyclohexyl-1-methoxycarbonylethyl ester (110 mg, 0.367 mmol) indioxane (1.5 ml). The mixture was stirred at ambient temperature for 1hour then diluted with water (10 ml). The aqueous layer was extractedwith diethyl ether (2×110 ml) which was discarded, then acidified topH=2 with 6M hydrochloric acid. The product was extracted into diethylether (2×10 ml), then the combined ethereal layers washed with saturatedaqueous sodium chloride (10 ml), dried (MgSO₄) and the solvent removedin vacuo to give morpholine-4-carboxylic acid1-carboxy-2-cyclohexylethyl ester as a white solid, yield 0.11 g, (100%from ester). TLC (single spot, Rf=0.20, methanol:dichloromethane 1:9),HPLC-MS (single main peak with Rt=7.614 mins, 286.2 [M+H]⁺, 287.2[M+2H]⁺, 593.3 [2M+Na]⁺).

[0320]_(δ)H (400 MHz, CDCl₃ at 298K), 0.75-1.00 (2H, CH ₂(cyclohexane),m), 1.02-1.28 (4H, CH ₂(cyclohexane), m), 1.33-1.46 (1H,CH(cyclohexane), m), 1.50-1.79 (6H, CH ₂(cyclohexane), m), 3.28-3.73(8H, CHOCH₂ and CH ₂NCH ₂, m), 4.92-5.02 (1Ha, m), 5.99 (1H, OH, brs).

EXAMPLE 9 (2S, 3S)2-Cyclohexylmethyl-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-4-morpholin-3yl-4-oxo-butyramide

[0321]

[0322] HPLC-MS 381.4 [M+H]⁺.

[0323] The required carboxylic acid building2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid (compound (22),Scheme 6) was prepared as follows:

[0324] (1) Preparation of2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid Methyl Ester.

[0325] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.HCl (1.12 g, 5.69mmol) then 1-hydroxybenzotriazole monohydrate (0.87 g, 5.69 mmol) wereadded to a stirred solution of 2R-(cyclohexylmethyl)succinic acid1-methyl ester (compound (21), 1.0 g, 4.38 mmol) in dimethylformamide(10 ml) at 0° C. under argon. The mixture was stirred for 25 minutesthen morpholine (0.7 ml, 8.76 mmol) was added drop-wise over 1 minuteand stirring continued at ambient temperature for 16 hours. The productwas extracted into ethyl acetate (200 ml) then washed with 1.0Mhydrochloric acid (3×100 ml), saturated aqueous sodium hydrogencarbonate solution (3×100 ml), water (100 ml), then saturated aqueoussodium chloride solution (100 ml1), dried (MgSO4), and the solventremoved in vacuo to give2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid methyl ester asan off-white solid, yield 1.22 g, (94%). HPLC-MS (single peak withRt=7.91 mins, 298.1 [M+H]⁺, 617.3 [2M+Na]⁺).

[0326] (2) Preparation of2R-cyclohexylmethyl-4-morpholinyl-4-oxo-butyric Acid (Compound (22),Scheme 6).

[0327] A solution of lithium hydroxide monohydrate (0.51 g, 12.18 mmol)in water (27 ml) was added a stirred solution of2R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid methyl ester(1.21 g, 4.06 mmol) in tetrahydrofuran (55 ml) and methanol (27 ml) at0° C. The mixture was stirred at ambient temperature for 1 hours thendiluted with water (100 ml). The aqueous layer was extracted withdiethyl ether (2×50 ml) which was discarded, then acidified to pH=1-2with 1M hydrochloric acid. The product was extracted intodichloromethane (3×50 ml), then the combined ethereal layers washed withwater (2×50 ml), saturated aqueous sodium chloride solution (2×50 ml),dried (MgSO₄) and the solvent removed in vacuo to leave a residue. Theresidue was purified by chromatography over silica gel eluting with agradient of methanol:dichloromethane 1:100→3:100. Appropriate fractionswere combined and the solvents removed in vacuo was to give2.R-cyclohexylmethyl-4-morpholin-4-yl-4-oxo-butyric acid (22) as a whitesolid, yield 0.82 g, (71%). HPLC-MS (single peak with Rt=6.769 mins,284.2 [M+H]⁺, 589.2 [2M+Na]⁺).

[0328]_(δ)H (400 MHz, CDCl₃ at 298K), 0.77-0.90 (2H, CH ₂(cyclohexane),m), 1.05-1.40 (4H, CH ₂(cyclohexane), m), 1.50-1.90 (7H,CH(cyclohexane), CH ₂(cyclohexane), m), 2.30-2.44 (2Hβ, m), 2.64-2.77(1Hα, m), 2.96-3.10 (1H, OH, brs), 3.40-3.78 (8H, CH ₂OCH ₂ and CH ₂NCH₂, m).

EXAMPLE 10 (2S, 3S) 2-Biphenyl-3-ylmethyl-pentanoic acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0329]

[0330] HPLC Rt=20.60 mins (>95%), HPLC-MS 366.1 [M+H]⁺, 753.2 [2M+Na]⁺.

[0331] The required carboxylic acid building block2RS-Biphenyl-3-yl-4-methylpentanoic acid (compound (25), Scheme 7) wasprepared as follows

[0332] (1) Preparation of Biphenyl-3-yl-acetic Acid Methyl Ester(Compound (24), Scheme 7)

[0333] Concentrated sulphuric acid (588 μL) was added to a solution of3-bromophenyl acetic acid (10 g, 46.5 mmol) in methanol (100 mL). Themixture was refluxed for 1.5 h and then cooled to ambient temperatureand evaporated under reduced pressure to afford a residue. The residuewas redissolved in diethyl ether (500 mL), washed with water (2×100 mL),brine (100 mL), dried (MgSO₄) and then evaporated under reduced pressureto afford 3-bromophenyl acetic acid methyl ester (10.65 g). The3-bromophenyl acetic acid methyl ester was dissolved in toluene (117 mL)then phenyl boronic acid (6.8 g, 55.69 mmol) added, followed by aaqueous solution of sodium carbonate (93 mL, 2M) andtetrakis(triphenylphosphine)palladium (1.6 g, 1.41 mmol). The mixturewas stirred overnight then cooled to ambient temperature and an aqueoussolution of saturated ammonium chloride (100 mL) added. The mixture wasextracted with ethyl acetate (2×200 mL), died (Na₂SO₄) and evaporatedunder reduced pressure to afford a residue. Flash chromatography of theresidue over silica (200 g) using ethyl acetate:heptane (3:48) as theeluent gave biphenyl-3-yl acetic acid methyl ester, yield 10.5 g, (99%),TLC (single Uw spot, R_(f)=0.24, 10% ethyl acetate in heptane),analytical HPLC Rt=19.55 min, HPLC-MS (single main UV peak withR_(t)=9.35 min, 227.1 [M+H]⁺).

[0334]_(δ)H (400 MHz, CDCl₃ at 298K) 3.76 (2H, s, CH₂CO₂CH₃), 3.77 (3H,s, OCH₃), 7.34-7.66 (9H, m, biphenyl-3-yl).

[0335] (2) Preparation of Biphenyl-3-yl-acetic Acid

[0336] Water (39 mL), followed by lithium hydroxide monohydrate (4.2 g,101.5 mmol) were added to a solution of biphenyl-3-yl acetic acid methylester (11.43 g, 50.57 mmol) in methanol (265 mL). The mixture wasstirred at ambient temperature for 2h then the organics were removedunder reduced pressure. The mixture was acidified with dilutehydrochloric acid (1M, 80 mL), extracted with chloroform (2×100 mL),dried (MgSO₄) and evaporated under reduced pressure to affordbiphenyl-3-yl acetic acid as a white solid, yield 10.6 g, (99%),analytical HPLC R_(t)=16.565 min, HPLC-MS (single main UV peak withR_(t)=7.91 min, 213.1 [M+H]⁺).

[0337]_(δ)H (400 MHz, CDCl₃ at 298K) 3.77 (2H, s, CH₂CO₂CH₃), 7.28-7.52(9H, m, biphenyl-3-yl).

[0338] (3) Preparation of 2RS-Biphenyl-3-yl-4-methylpentenoic Acid

[0339] A solution of biphenyl-3-yl acetic acid (7.0 g, 33 mmol) inanhydrous tetrahydrofuran (84 mL) was added dropwise to a solution oflithium diisopropyl amide (36.4 mL, 2M solution in hexanes) in anhydroustetrahydrofuran (84 mL) at −78° C. The mixture was allowed to warm to 0°C. and stirred for 40 min. The mixture was then cooled to −78° C. and3-bromo-2-methylpropene (4.97 mL) rapidly added. The mixture was stirredfor 1 h at −78° C. then water (28 mL) added and the organics removedunder reduced pressure. The mixture was then acidified with hydrochloricacid (6M, 14 ml), extracted with ethyl acetate (3×100 ml), dried (MgSO4)and evaporated under reduced pressure to afford a residue. Flashchromatography of the residue over silica (400 g) usingmethanol:dichloromethane (3:97) as the eluent afforded impure2-biphenyl-3-yl-4-methylpent-4-enoic acid (8.3 g). Flash chromatographyover silica (400 g) using methanol:dichloromethane (1.5:98.5) affordedpure 2-biphenyl-3-yl-4-methylpent-4-enoic acid, yield 5.27 g, (60%), TLC(single UV spot, R_(f)=0.28, 5% methanol in dichloromethane), analyticalHPLC R_(t)=19.99 min, HPLC-MS (single main UV peak with R_(t)=9.57 min,267.1 [M+H]⁺).

[0340]_(δ)H (400 MHz, CDCl₃ at 298K), 1.765 (3H, s, CH₃), 2.53 (1H, dd,J=6.6 and 14.7 Hz, 3-HI), 2.91 (1H, dd, J=8.9 and 14.7 Hz, 3-HI), 3.92(1H, dd, J=6.6 and 8.9 Hz, 2H), 4.79 (2H, d, J=10.7 Hz, 5-H₂), 7.30-7.62(9H, m, biphenyl-3-yl).

[0341] (4) Preparation of 2RS-Biphenyl-3-yl-4-methylpentanoic Acid(Compound (25), Scheme 7)

[0342] Palladium on carbon (10%, 300 mg) was added portionwise to asolution of 2RS-biphenyl-3-ylmethylpent-4-enoic acid (1 g, 3.76 mmol) inethanol (40 mL) at 0° C. A hydrogen atmosphere was then introduced andthe mixture allowed to warm to ambient temperature. The mixture wasstirred for 18h, then the hydrogen atmosphere removed and the mixturefiltered over Celite and the catalyst washed with ethanol (40 mL). Thecombined organic filtrate was concentrated under reduced pressure toafford a residue, which was flash chromatographed over silica (150 g)using methanol:dichloromethane (1:99) as the eluent to afford2RS-biphenyl-3-yl-4-methylpentanoic acid, (25) yield 980 mg, (98%), TLC(single UV spot, R_(f)=0.45, 5% methanol in dichloromethane), analyticalHPLC R_(t)=20.92 min, HPLC-MS (single main UV peak with R_(t)=10.15 min,269.1 [M+H]⁺, 291.1 [M+Na]⁺). 8H (400 MHz, CDCl₃ at 298K), 0.93 (6H, d,J=6.6 Hz, 2×CH₃), 1.52-1.57 (1H, m, 4-H₁), 1.71-1.76 (1H, m, 3-H₁),1.97-2.05 (1H, m, 3-H₁), 3.66 (1H, t, J=7.8 Hz, 2-H₁), 7.32-7.60 (9H, m,biphenyl-3-yl).

EXAMPLE 11 (2R, 3s)2-Benzyloxy-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-3-phenyl-propionamide

[0343]

[0344] HPLC Rt=18.35 mins (>90%), HPLC-MS 354.2 [M+H]⁺, 729.3 [2M+Na]⁺.

[0345] Following the general details from Scheme 1, the required bicyclebuilding block (2R, 3S) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamicacid 9H-fluoren-9-ylmethyl ester (6b) was prepared as follows:

[0346] (1) Preparation of (1S, 1′R)[3-diazo-1-(1-tert-butoxyethyl)-2-oxo-propyl]carbamic acid9H-fluoren-9-ylmethyl Ester.

[0347] A solution of iso-butyl chloroformate (1.05 g, 7.7 mmol) indichloromethane (14 ml) and a solution of 4-methylmorpholine (1.42 g, 14mmol) in dichloromethane (14 ml) were simultaneously added to a stirredsuspension of Fmoc-O-(tert-butyl ether)-L-threonine (2.78 g, 7 mmol) indichloromethane (70 ml) at −15° C. over 10 minutes under an atmosphereof nitrogen. Ethereal diazomethane [generated from diazald (7.14 g, ˜21mmol) addition in diethyl ether (115 ml) to sodium hydroxide (8.022 g)in water (11 ml)/ethanol (31 ml) at 60° C.] was then cautiously addedand the resulting yellow solution was stirred at room temperature for 90minutes. Acetic acid (3 ml) was cautiously added (until effervescencehad ceased) then the mixture was diluted with tert-butyl methyl ether(70 ml). The ethereal layer was washed with water (3×70 ml), dried(Na₂SO₄) and the solvent removed in vacuo to leave (1S, 1′R)[3-diazo-1-(1-tert-butoxyethyl)-2-oxo-propyl]carbamic acid9H-fluoren-9-ylmethyl ester as a yellow solid (4.28 g) which was usedwithout further purification.

[0348] (2) Cyclisation to (ZR, 3S)(2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl Ester.

[0349] A solution of lithium chloride (2.97 g, 70 mmol) in 80% aceticacid in water (105 ml) was added to (1S, 1′R)[3-diazo-1-(1-tert-butoxyethyl)-2-oxo-propyl]carbamic acid9H-fluoren-9-ylmethyl ester (4.28 g, 7 mmol). The yellow oily suspensionwas stirred for 2 hours whereupon a pale yellow solution formedaccompanied by the evolution of a gas. The solvents were removed invacuo then the residue was dissolved in ethyl acetate (42 ml), washedwith 10% aqueous sodium carbonate solution (2×42 ml) and saturatedaqueous sodium chloride solution (42 ml). The combined aqueous layerswere extracted with ethyl acetate (2×42 ml) then the combined ethylacetate layers were dried (Na₂SO₄) and the solvents removed in vacuo toleave a yellow gum (3.44 g). The yellow gum was purified bychromatography over silica gel eluting with a gradient of n-heptane:ethyl acetate 9:1→7:3. Appropriate fractions were combined and thesolvents removed in vacuo to leave (2R, 35)(2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester as a white crystalline solid (2.92 g, 62%from starting acid). TLC (single WV spot, Rf=0.27, n-heptane: ethylacetate 2:1), analytical HPLC peak Rt=17.96 mins, HPLC-MS (single UVpeak with Rt=8.48 mins, 360.0 [M+Na]⁺, 361.0 [M+H+Na]⁺).

[0350]_(δ)H (CDCl₃ at 298K); 1.37-1.60 (3H, CH ₃CHO, brs), 3.68-3.86(1H, CH₃CHO m), 3.89-4.09 (2H, Fmoc CH ₂, m), 4.16-4.34 (2H, Fmoc H-9and NHCHCO, m), 4.36-4.57 (2H, OCH ₂CO, dd), 5.05-5.25 (1H, NH, brs),7.28-7.47 (4H, ArH, Fmoc H-2 and H-7, ArH, Fmoc H-3 and H-6), 7.50-7.66(2H, ArH, Fmoc H-1 and H-8), 7.73-7.85 (2H, ArH, Fmoc HA and H-5).

[0351]_(δ)C (CDCl₃ at 298K); 18.61 (u, CH₃CHO), 46.69 (u, Fmoc C-9),62.26 (u, NHCHCO), 66.82 (d, Fmoc CH₂), 70.38 (d, COCH₂), 76.93 (u, CH₃CHO), 119.60 (u, Fmoc C-4 and C-5), 124.55 (u, Fmoc C-1 and C-8), 126.69(u, Fmoc C-2 and C-7), 127.37 (u, Fmoc C-3 and C-6), 140.92 (q, Fmoc C4′and C-5′), 143.15/143.20 (q, Fmoc C-1′ and C-8′), 155.61 (q, OCON),211.38 (q, COCH₂).

[0352] Following the general details from Scheme 2, the required bicyclebuilding (2R, 3S) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6b) was converted to building block-linkerconstruct (8b) as follows:

[0353] (2R, 3S) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6b) (354 mg, 1.05 mmol, 1 eq) was dissolvedin a mixture of ethanol (9.6 mL) and water (1.4 mL) containing sodiumacetate.trihydrate (218 mg, 1.6 mmol, 1.5 eq).4-[[(hydrazinocarbonyl)amino] methyl]cyclohexanecarboxylic acid.trifluoroacetate (346 mg, 1.05 mmol, 1 eq, Murphy, A. M. et al, J. Am.Chem. Soc., 114, 3156-3157, 1992) was added and the mixture refluxed for2 hr. Chloroform (100 mL) was added and the organics washed with diluteaqueous hydrochloric acid (0.1 M, 1×100 mL). The acidic layer wasbackwashed with chloroform (3×100 mL) and the combined organics washedwith brine (100 mL), dried (Na₂SO₄) and evaporated under reducedpressure to afford linker construct (8b) as a white solid (660 mg).Analytical HPLC indicated one main peak at R_(t)=17.011 min, HPLC-MS(main UW peak with R_(t)=7.96 min, 535.3 [M+H]⁺. Crude (8b) was useddirectly for construct loading.

[0354] Following the general details from Scheme 2, the requiredbuilding block-linker construct (8b) was attached to the solid phaseproviding loaded building block-linker construct (9b) as follows:

[0355] Building block-linker construct (8b) (1.0 mmole),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro phosphate(HBTU, 379 mg, 1.0 mmole), 1-hydroxybenzotriazole.hydrate and (HOBT, 153mg, 1.0 mmole) were dissolved in dimethylformamide (5 mL) andN-methylmorpholine (NMM, 220 μL, 2.0 mmole) added. After pre-activationfor 5 minutes, free amine gears (250×1.2 μmole) were added, followed bydimethylformamide (30 mL) and left overnight. The spent couplingsolution was then added to free amine crowns (25×10 μmole) and leftovernight. Standard washing and analyses indicated loading at >95%.

[0356] 2RS-benzyloxy-3-phenylpropionic acid was coupled under standardconditions to loaded building block-linker construct (9b) (followingstandard removal of Fmoc), then cleaved to provide EXAMPLE 11. The crudeexample was analysed (see general techniques). HPLC Rt=18.35 mins(>90%), HPLC-MS 354.2 [M+H]⁺, 729.3 [2M+Na]⁺.

[0357] The following examples (12-23) were prepared as detailed forEXAMPLE 11, coupling with the required reagents to provide the fulllength molecule.

EXAMPLE 12 (2R, 3s) 4-Methyl-2-(4trifluoromethylbenzyloxy)-pentanoicAcid (2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0358]

[0359] HPLC Rt=20.83 nmins (>90%), BPLC-MS 388.2 [M+H]⁺, 797.3 [2M+Na]⁺.

[0360] The required carboxylic acid building block2RSA4-methyl-2-(4-trifluoromethyl-benzyloxy)-pentanoic acid was preparedas follows

[0361] (1) Preparation of2RS-4-methyl-2-(4-trifluoromethyl-benzyloxy)-pentanoic Acid

[0362] Sodium hydride (333 mg of 60% dispersion in oil, 8.3 mmol) wasadded in two portions to a stirred mixture of(S)-2-hydroxy-4-methylpentanoic acid (0.5 g, 3.8 mmol),dimethylformamide (5 ml) and dichloromethane (5 ml) at 0° C. over 5minutes. The mixture was stirred at 0° C. for 5 minutes then at ambienttemperature for 45 minutes. 4-(trifluoromethyl)benzyl bromide (0.73 ml,4.7 mmol) was added then the mixture stirred for 5 hours before addingpotassium iodide (50 mg, 0.3 mmol) and dimethylformamide (5 ml). Themixture was stirred for 20 hours then heated at 55° C. for 1 hour thenallowed to cool to ambient temperature and poured into water (15 ml). Asaturated aqueous sodium chloride (5 ml) was added then the mixture wasextracted with dichloromethane (5 ml then 10 ml) that was discarded. Theaqueous layer was acidified using 1M hydrochloric acid (10 ml) thenextracted with dichloromethane (2×10 ml). The dichloromethane layer wasdried (MgSO₄) and the solvent removed in vacuo. The residue (0.52 g) wasdissolved in dimethylformamide (5 ml) then cooled to 0° C. before addingsodium hydride (160 mg of 60% dispersion in oil, 4 mmol). The mixturewas stirred for 30 minutes then polymer bound isocyanate (320 mg, 2 mmolNg⁻¹) added. The mixture was stirred for 2 hours at ambient temperaturethen poured into water (15 ml). 1M Hydrochloric acid (10 ml) was addedthen the product was extracted into dichloromethane (2×10 ml), dried(Na₂SO₄) and the solvent removed in vacuo. The residue was purified byflash chromatography over silica gel eluting with a gradient ofmethanol: dichloromethane 0:1→1:20. Appropriate fractions were combinedand the solvents removed in vacuo to leave2RS-4-Methyl-2-(4-trifluoromethylbenzyloxy)-pentanoic acid, yield 121 mg(11%) as a colourless oil. HPLC-MS (single peak with Rt=9.66 mins, 291.1[M+H]⁺, 313.1 [M+Na]⁺).

EXAMPLE 13 (2R, 3S)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0363]

[0364] HPLC Rt=19.73 mins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0365] Prepared as detailed for EXAMPLE 3, but using loaded buildingblock-linker construct (9b)

EXAMPLE 14 (2R, 3S)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0366]

[0367] HPLC Rt=19.73 mins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0368] Prepared as detailed for EXAMPLE 4, but using loaded buildingblock-linker construct (9b)

EXAMPLE 15 (2R, 3) 2-(4-tert-Butyl-benzylsulfanyl)+methyl-pentanoic Acid(2-methyl-4-oxo-tetrahydro-furan-3-yl)-amide

[0369]

[0370] HPLC Rt=23.21 mins (>95%), HPLC-MS 392.2 [M+H]⁺, 805.4 [2M+Na]⁺.

[0371] The required carboxylic acid building block2S-(4-tert-Butyl-benzylsulfanyl)-4-methyl-pentanoic acid (analogue ofcompound (16), Scheme 4) was prepared from (R)-2-bromo-4-methylpentanoicacid as follows:

[0372] (1) Preparation of 2R-Bromo-4-methylpentanoic Acid

[0373] A solution of sodium nitrite (5.1 g, 73 mmol) in water (15 ml)was added drop-wise at 0° C. over 5 hours to a stirred mixture ofD-leucine (8.75 g, 67 mmol), potassium bromide (29.75 g, 0.25 mol) andconcentrated sulphuric acid (8.6 ml) in water (100 ml). The mixture wasstirred for 30 minutes at 0° C. then at ambient temperature for 20hours. The product was extracted into diethyl ether (2×150 ml) then thecombined ethereal layers were washed with saturated aqueous sodiumchloride solution (2×100 ml), dried (MgSO₄) and the solvent removed invacuo. The residue was purified by flash chromatography over silica geleluting with a gradient of methanol: dichloromethane 1:50→1:20.Appropriate fractions were combined and the solvents removed in vacuo toleave 2R-bromo-4-methylpentanoic acid as a colourless oil, yield 1.60 g,(12.3%). TLC (single spot, Rf=0.2, methanol: dichloromethane 1:20).Additionally, a second crop (5.2 g, 40%) of slightly impure product wasobtained

[0374]_(δ)H (400z CDCl₃ at 298K), 0.95 and 0.99 (both 3H, CH ₃CH, d,J=6.55 Hz), 1.77-1.89 (1H, CH₃CH, m), 1.93 (2Hβ, m), 4.31 (1Hα, t, J=7.7Hz), 9.3 (1H, CO₂ H, brs).

[0375] (2) Preparation of2S-(4tert-butylbenzylsulfanyl)-4-methylpentanoic Acid (Analogue ofCompound (16), Scheme 4)

[0376] A solution of 2R-bromo-4-methylpentanoic acid (1.1 g, 5.6 mmol)and (4-(tert-butyl)phenyl)methanethiol (1.0 g, 5.6 mmol) indimethylformamide (15 ml) was purged with nitrogen for 5 minutes thencooled to 0° C. Triethylamine (0.79 ml, 5.7 mmol) was added drop-wiseover 1 minute then the mixture was stirred for two days at ambienttemperature. The solvents were removed in vacuo and residue purified byflash chromatography over silica gel eluting with a gradient ofmethanol: dichloromethane 0:1→1:20. Appropriate fractions were combinedand the solvents removed in vacuo to leave a residue which was purifiedby flash chromatography over silica gel eluting with ethylacetate:heptane 2:5. Appropriate fractions were combined and thesolvents removed in vacuo to give2S-(4-tert-butylbenzylsulfanyl)-4-methylpentanoic acid as a colourlessoil, yield 150 mg, (9%). TLC (single spot, Rf=0.2, heptane: ethylacetate 5:2), analytical HPLC with main peak Rt=22.117 mins, HPLC-MS(main V peak with Rt=11.072 mins, 317.2 [M+Na]⁺).

[0377]_(δ)H (400 MHz, CDCl₃ at 298K), 0.70 and 0.85 (both 3H, CH ₃CH, d,J=6.3), 1.29 (9H, (CH ₃)₃C, s), 1.44-1.51 (1H, CH₃CH, m), 1.62-1.75(2Hβ, m), 3.15-3.20 (1Hα, m), 3.81 and 3.88 (both 1H, SCH ₂, d, J=13.2Hz), 7.25-7.35 (4H, aromatic).

EXAMPLE 16 (2R, 3S)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4oxo-tetrahydrofuran-3-yl)-propionamide

[0378]

[0379] HPLC Rt=17.65 mins (>80%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0380] Prepared as detailed for EXAMPLE 5, but using loaded buildingblock-linker construct (9b)

EXAMPLE 17 (2R, 3S)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0381]

[0382] HPLC Rt=17.61 mins (>95%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0383] Prepared as detailed for EXAMPLE 6, but using loaded buildingblock-linker construct (9b)

EXAMPLE 18 (2R, 3S)2-(4-tert-Butyl-phenylmethanesulfonyl)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0384]

[0385] HPLC Rt=21.19 mins (>95%), HPLC-MS 424.2 [M+H]⁺, 869.4 [2M+Na]⁺.

[0386] Prepared as detailed for EXAMPLE 7, but using loaded buildingblock-linker construct (9b)

EXAMPLE 19 (2R, 3s) Morpholin-4-carboxylic acid2-cyclohexyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylEster

[0387]

[0388] HPLC-MS 383.4 [M+H]⁺.

[0389] Prepared as detailed for EXAMPLE 8, but using loaded buildingblock-linker construct (9b)

EXAMPLE 20 (2R, 3s)2-Cyclohexylmethyl-N-(2-methyl-4oxo-tetrahydrofuran-3-yl)-4-morpholin-4-yl-4-oxo-butyramide

[0390]

[0391] HPLC-MS 381.4 [M+H]⁺.

[0392] Prepared as detailed for EXAMPLE 9, but using loaded buildingblock-linker construct (9b)

EXAMPLE 21 (2R, 3S) 2-Biphenyl-3-yl-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0393]

[0394] HPLC Rt=20.62 mins (>95%), HPLC-MS 366.1 [M+H]⁺, 753;2 [2M+Na]⁺.

[0395] Prepared as detailed for EXAMPLE 10, but using loaded buildingblock-linker construct (9b)

EXAMPLE 22 (2R, 3s)4-Methyl-2-[2-oxo-2-(3-phenyl-pyrrol-1-yl)-ethyl]-pentanoic Acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0396]

[0397] HPLC Rt=19.80 & 20.16 mins (19.9 & 80.1%), HPLC-MS 397.2 [M+H]⁺,815.4 [2M+Na]⁺.

[0398] The required carboxylic acid building block (2R)4-Methyl-2-[2-oxo-2-(3-phenyl-pyrrol-1-yl)-ethyl]-pentanoic acid(analogue of compound (22), Scheme 6) was as follows:

[0399] (1) Preparation of 4-Phenyl-1H-pyrrole-3-carboxylic Acid MethylEster

[0400] A solution of 4-toluenesulphonylmethyl isocyanide (4.9 g, 25.2mmol) and methyl trans-cinnamate (4.0 g, 24.7 mmol) in diethyl ether (40ml) and dimethyl sulphoxide (20 ml) was added drop-wise over 30 minutesto a stirred suspension of sodium hydride (60% dispersion, 1.25 g, 31.3mmol) in diethyl ether (100 ml) under nitrogen. After 20 minutes afurther portion of dimethyl sulphoxide (25 ml) was added to the reactionvessel. The mixture was stirred for 1 hour then water (200 ml) wascautiously added. The product was extracted into dichloromethane (1×400ml and 1×250 ml) then the combined dichloromethane layers were dried(Na₂SO₄) and the solvent removed in vacuo. The residue was purified byflash chromatography over silica gel eluting with a gradient ofheptane:ethyl acetate:dichloromethane 2:1:0→1:1:1. Appropriate fractionswere combined and the solvents removed in vacuo to leave4-phenyl-1H-pyrrole-3-carboxylic acid methyl ester as a white solid(1.55 g, 31%). TLC (single spot, Rf=0.25, heptane:ethyl acetate 2:1),HPLC-MS (main UV peak with Rt=7.849 mins, 202.1 [M+H]⁺, 425.1 [2M+Na]⁺).

[0401] (2) Preparation of 3-Phenyl-1H-pyrrole

[0402] A solution of potassium hydroxide (4.3 g, 77 mmol) in water (40ml) was added to a suspension of 4-phenyl-1H-pyrrole-3-carboxylic acidmethyl ester (1.55 g, 7.7 mmol) in methanol (40 ml). The mixture washeated at reflux for 2.5 hours then the majority of solvents removedfrom the resulting solution by distillation in vacuo. The residue wasdiluted with water (100 ml) and ice (30 ml) added then the mixture wasacidified with hydrochloric acid (1M) to pH=1. The precipitate wascollected by filtration in vacuo then washed with water (75 ml),dichloromethane (75 ml), water (20 ml) then dichlororomethane (75 ml).The white solid was dried in vacuo then heated with ethanolamine (10 ml)at 190° C. under an atmosphere of nitrogen for 2.25 hours. The mixturewas allowed to cool to ambient temperature then poured onto ice (150ml). Water (50 ml) was added then the product was extracted intodichloromethane (3×50 ml). The dichloromethane layers were combined thendried (Na₂SO₄) and the solvent removed in vacuo to leave an oil (900 mg)which was purified by flash chromatography over silica gel eluting withethyl acetate:heptane 1:5. Appropriate fractions were combined and thesolvents removed in vacuo to leave 3-phenyl-1H-pyrrole as a yellow-greenoil which solidified after storage at −80° C. for 20 hours (810 mg,74%). TLC (single spot, Rf=0.25, heptane:ethyl acetate 5:1), HPLC-MS(single UV peak with Rt=8.493 mins, 144.1 [M+H]⁺).

[0403]_(δ)H (CDCl₃ at 298K); 6.54, 6.80 and 7.06 (each 1H, pyrrole, m),7.15-7.51 (5H, aromatic), 8.22 (1H, NH, brs)

[0404] (3) Preparation of (2R) Isobutyl-succinic Acid

[0405] A solution of lithium hydroxide monohydrate (26 mg, 0.61 mmol) inwater (2501 μl) was added to a stirred solution of (2R)2-isobutylsuccinic acid 1-methyl ester (100 mg, 0.53 mmol) in1,4-dioxane (500 μl). The mixture was stirred for 2 hours then lithiumhydroxide monohydrate (26 mg, 0.61 mmol) was added and stirringcontinued for 24 hours. The majority of solvents were removed bydistillation in vacuo then the residue was dissolved in water (5 ml) andacidified using hydrochloric acid (1M) to pH=1. The product wasextracted into dichloromethane (5×5 ml), dried (Na₂SO₄) and the solventremoved in vacuo to obtain (2R) 2-isobutylsuccinic acid (49 mg, 53%) asa solid. HPLC-MS (single UV peak with Rt=6.223 mins, 197.1 [M+Na]⁺).

[0406] (4) Preparation of (2R)4-Methyl-2-[2-oxo-2-(3-phenyl-pyrrol-1-yl)-ethyl]-pentanoic Acid

[0407] A solution of (2R)-2-isobutylsuccinic acid (35 mg, 0.2 mmol) inacetyl chloride (1.2 ml) was heated at reflux under nitrogen for 2.2hours then volatile components removed by distillation in vacuo to leave(3R)-3-isobutyldihydrofuran-2,5-dione as an oily residue which was usedwithout further purification.

[0408] Sodium hydride (12 mg of 60% dispersion in oil, 0.3 mmol) wasadded to a stirred solution of 3-phenylpyrrole (43 mg, 0.30 mmol) intetrahydrofuran (1.0 ml) under nitrogen. The mixture was stirred for 15minutes then added via cannula to a stirred solution of the(3R)-3-isobutyldihydrofuran-2,5-dione (0.2 mmol) in tetrahydrofuran (1.0ml) at 0° C., then stirred for 5 hours. The majority of solvents wereremoved in vacuo then water (10 ml) added. The aqueous layer wasextracted with ethyl acetate (5 ml) then saturated aqueous sodiumchloride solution (5 ml) added and the aqueous layer extracted with anadditional portion of ethyl acetate (5 ml). The aqueous layer wasacidified using hydrochloric acid (1M) to pH=1 then the productextracted into ethyl acetate (2×5 ml), dried (MgSO₄) and the solventremoved in vacuo. The residue was purified by flash chromatography oversilica gel eluting with a gradient of methanol:dichloromethane1:99→3:17. Appropriate fractions were combined and the solvents removedin vacuo to leave a mixture (approximately 1:1) of(2R)-4-methyl-2-[oxo-2-(3-phenylpyrrol-1-yl)ethyl]pentanoic acid and(38)-5-Methyl-3-(3-phenyl-pyrrole-1-carbonyl)-hexanoic acid (1.6 mg, 2%)as an oil. TLC (two spots, Rf=0.8 and Rf=0.75, methanol:dichloromethane1:9), analytical HPLC two unresolved UV peaks with Rt=19.020 and 19.127mins, HPLC-MS (two unresolved UV peaks with Rt=9.724 and 9.861 mins,300.2 [M+H]⁺, 621.3 [2M+Na]⁺).

[0409] This mixture was used directly for the preparation of EXAMPLE 22.

EXAMPLE 23 (2R, 3S) 2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]4methyl-pentanoic acid (2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0410]

[0411] HPLC Rt=16.03 & 16.53 mins (>80%), HPLC-MS 387.2 [M+H]⁺, 795.4[2M+Na]⁺.

[0412] The required carboxylic acid building block (2R)2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]-4-methyl-pentanoicacid (analogue of compound (22), Scheme 6) was as follows:

[0413] (1) Preparation of2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]-4-methyl-pentanoicAcid Methyl Ester.

[0414] iso-Butyl chloroformate (33.64 mg, 0.2463 mmol) indichloromethane (2 ml) and N-methyl morpholine (49.83 mg, 0.4927 mmol)in dichloromethane (2 ml) were simultaneously added to a stirredsolution of (R)-2-isobutyl succinic acid 1-methyl ester (42.15 mg,0.2239 mmol) in dichloromethane (2 ml) at −15° C. under argon over 5minutes. The mixture was stirred at −15° C. for 15 minutes. A freshlyprepared solution of 1,2,3,4-tetrahydro-isoquinoline (29.83 mg, 0.2239mmol) in dichloromethane (2 ml) and N-methylmorpholine (24.92 mg, 0.2463mmol) was then added dropwise. The mixture was then stirred at ambienttemperature for 16 hours. The reaction mixture was poured into saturatedaqueous sodium chloride solution (1 ml). The aqueous layer was extractedwith diethyl ether (2×5 ml) which was discarded. The aqueous layer wasacidified with 1M hydrochloric acid to pH (12), then the product wasextracted into dichloromethane (3×5 ml). The combined dichloromethanelayers were washed with water (2×5 ml), saturated aqueous sodiumchloride solution (2×5 ml) then dried (Na₂SO₄). The solvent was removedin vacuo to give a residue which was purified over silica gel elutingwith a gradient of n-heptane:ethyl acetate 4:1. Desired fractions werecombined and reduced in vacuo to leave2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]-4-methyl-pentanoicacid methyl ester as a colourless oil (35 mg, 51.5% from starting acid).TLC (single UV spot, Rf=0.6, n-heptane:ethyl acetate 1:1), HPLC-MS(single UV peak with Rt=9.544 mins, 304.2 [M+H]⁺, 305.2 [M+2]⁺, 306.2[M+3]⁺, 629.3 [2M+Na]⁺.

[0415] (2) Preparation of2-[2-(3,4-dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]4-methyl-pentanoicAcid.

[0416] A solution of lithium hydroxide monohydrate (0.01452 g, 0.3461mmol) in water (1.5 ml) was added drop-wise over 1 minute to a solutionof 2-[2-(3,4-dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]4-methyl-pentanoicacid methyl ester (0.035 g, 0.1154 mmol) in tetrahydrofuran:methanol 2:1(9 ml) at 0° C. The mixture was stirred at ambient temperature for 16hours then poured into saturated aqueous sodium chloride solution (10ml). The aqueous layer was extracted with diethyl ether (2×10 ml) whichwas discarded. The aqueous layer was acidified with 1M hydrochloric acidto pH (12), then the product was extracted into dichloromethane (3×10ml). The combined dichloromethane layers were washed with water (2×10ml), saturated aqueous sodium chloride solution (2×10 ml) then dried(Na₂SO₄) and the solvents removed in vacuo to leave2-[2-(3,4-dihydro-1H-isoquinolin-2-yl)-2-oxo-ethyl]-4-methyl-pentanoicacid as a colourless oil (4.0 mg, 12%). TLC (main spot, Rf=0.2,methanol:dichloromethane 1:10), analytical HPLC Rt=13.297 min. HPLC-MS(290.2 [M+I]+, 312.2 [M+Na]⁺, 601.3 [2M+Na]⁺).

EXAMPLE 24 (2S, 3R)2-Benzyloxy-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-3-phenyl-propionamide

[0417]

[0418] HPLC Rt=18.34 mins (>90%), HPLC-MS 354.2 [M+H]⁺.

[0419] Following the general details from Scheme 1, the required bicyclebuilding block (25, 3R) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamicacid 9H-fluoren-9-ylmethyl ester (6c) was prepared as follows:

[0420] (1) Preparation of (1R, 1′S)[3-diazo-1-(1-hydroxyethyl)₂-oxo-propyl]carbamic Acid9H-fluoren-9-ylmethyl Ester.

[0421] A solution of iso-butyl chloroformate (1.05 g, 7.7 mmol) indichloromethane (7 ml) and a solution of 4-methylmorpholine (1.42 g, 14mmol) in dichloromethane (7 ml) were simultaneously added to a stirredsuspension of Fmoc-D-threonine (2.39 g, 7 mmol) in dichloromethane (70ml) at −15° C. over 10 minutes under an atmosphere of nitrogen. Etherealdiazomethane [generated from diazald (7.14 g, ˜21 mmol) addition indiethyl ether (115 ml) to sodium hydroxide (8.022 g) in water (11ml)/ethanol (31 ml) at 60° C.] was then cautiously added and theresulting yellow solution was stirred at room temperature for 90minutes. Acetic acid (3 ml) was cautiously added (until effervescencehad ceased) then the mixture was diluted with tert-butyl methyl ether(70 ml). The ethereal layer was washed with water (3×70 ml), dried(Na₂SO₄) and the solvent removed in vacuo to leave (1R, 1′S)[3-diazo-1-(1-hydroxyethyl)-2-oxo-propyl]carbamic acid9H-fluoren-9-ylmethyl ester as a yellow solid (3.43 g) which was usedwithout further purification.

[0422] (2) Cyclisation to (2S, 3R)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic Acid 9H-fluoren-9-ylmethylester.

[0423] A solution of lithium chloride (2.97 g, 70 mmol) in 80% aceticacid in water (105 ml) was added to (1R, 1′S)[3-diazo-1-(1-hydroxyethyl)-2-oxo-propyl]carbamic acid9H-fluoren-9-ylmethyl ester (3.43 g, ˜7 mmol). The yellow oilysuspension was stirred for 2 hours whereupon a pale yellow solutionformed accompanied by the evolution of a gas. The solvents were removedin vacuo then the residue was dissolved in ethyl acetate (42 ml), washedwith 10% aqueous sodium carbonate solution (2×42 ml) and saturatedaqueous sodium chloride solution (42 ml). The combined aqueous layerswere extracted with ethyl acetate (2×42 ml) then the combined ethylacetate layers were dried (Na₂SO₄) and the solvents removed in vacuo toleave a yellow gum (2.26 g). The yellow gum was purified bychromatography over silica gel eluting with a gradient ofn-heptane:ethyl acetate 9:1→7:3. Appropriate fractions were combined andthe solvents removed in vacuo to leave (2S, 3R)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic acid 9H-fluoren-9-ylmethylester as a white crystalline solid (659 mg, 28% from starting acid). TLC(single UV spot, Rf=0.27, n-heptane:ethyl acetate 2:1), analytical HPLCpeak Rt=18.02 mins, HPLC-MS (single UV peak with Rt=8.47 mins, 338.1[M+H]⁺, 360.1 [M+Na]⁺, 361.0 [M+H+Na]⁺.

[0424]_(δ)H (CDCl₃ at 298K); 1.39-1.60 (3H, CH ₃CHO, brs), 3.72-3.85(1H, CH₃CHO m), 3.88-4.09 (2H, Fmoc CH ₂, m), 4.15-4.33 (2H, Fmoc H-9and NHCHCO, m), 4.39-4.58 (2H, OCH ₂CO, dd), 5.07-5.25 (1H, NH, brs),7.28-7.38 (2H, ArH, Fmoc H-2 and H-7), 7.39-7.47 (2H, ArH, Fmoc H-3 andH-6), 7.52-7.66 (2H, ArH, Fmoc H-1 and H-8), 7.72-7.86 (2H, ArH, FmocH-4 and H-5).

[0425]_(δ)C (CDCl₃ at 298K); 19.45 (u, CH₃CHO), 47.52 (u, Fmoc C-9),63.09 (u, NHCHCO), 67.65 (d, Fmoc CH₂), 71.21 (d, COCH₂), 77.80 (u, CH₃CHO), 120.43 (u, Fmoc C-4 and C-5), 125.38 (u, Fmoc C-1 and C-8), 127.51(u, Fmoc C-2 and C-7), 128.20 (u, Fmoc C-3 and C-6), 141.75 (q, FmocC-4′ and C-5′), 143.98/144.03 (q, Fmoc C-1′ and C-8′), 156.45 (OCON),212.21(COCH₂).

[0426] Following the general details from Scheme 2, the required bicyclebuilding (2S, 3R) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6c) was converted to building block-linkerconstruct (8c) as follows:

[0427] (2S, 3R) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6c) (354 mg, 1.05 mmol, 1 eq) was dissolvedin a mixture of ethanol (9.6 mL) and water (1.4 mL) containing sodiumacetate.trihydrate (218 mg, 1.6 mmol, 1.5 eq).4-[[(hydrazinocarbonyl)amino] methyl]cyclohexanecarboxylic acid.trifluoroacetate (346 mg, 1.05 mmol, 1 eq, Murphy, A. M. et al, J. Am.Chem. Soc., 114, 3156-3157, 1992) was added and the mixture refluxed for2 hr. Chloroform (100 mL) was added and the organics washed with diluteaqueous hydrochloric acid (0.1 M, 1×100 mL). The acidic layer wasbackwashed with chloroform (3×100 mL) and the combined organics washedwith brine (100 mL), dried (Na₂SO₄) and evaporated under reducedpressure to afford linker construct (8c) as a white solid (660 mg).Analytical HPLC indicated one main peak at R_(t)=17.14 min, HPLC-MS(main UV peak with R_(t)=7.91 min, 535.3 [M+H]⁺. Crude (8c) was useddirectly for construct loading.

[0428] Following the general details from Scheme 2, the requiredbuilding block-linker construct (8c) was attached to the solid phaseproviding loaded building block-linker construct (9c) as follows:

[0429] Building block-linker construct (8c) (1.0 mmole),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro phosphate(HBTU, 379 mg, 1.0 mmole), 1-hydroxybenzotriazole.hydrate and HOBT, 153mg, 1.0 mmole) were dissolved in dimethylformamide (5 mL) andN-methylmorpholine (NMM, 220 μL, 2.0 mmole) added. After pre-activationfor 5 minutes, free amine gears (250×1.2 μmole) were added, followed bydimethylformamide (30 mL) and left overnight. The spent couplingsolution was then added to free amine crowns (25×10 μmole) and leftovernight. Standard washing and analyses indicated loading at >95%.

[0430] Following the general details from Scheme 2, the required loadedbuilding block-linker construct (9c) was elaborated on the solid phaseas follows:

[0431] 2RS-benzyloxy-3-phenylpropionic acid was coupled under standardconditions to loaded building block-linker construct (9c) (followingstandard removal of Fmoc), then cleaved to provide EXAMPLE 24. The crudeexample was analysed (see general techniques). HPLC Rt=18.34 mins(>90%), HPLC-MS 354.2 [M+H]⁺.

[0432] The following examples (25-34) were prepared as detailed forEXAMPLE 24, coupling with the required reagents to provide the fulllength molecule.

EXAMPLE 25 (2S, 3R)N-(2-Methyloloxo-tetrahydrofuran-3-yl)-3-phenyl-2-(4-trifluoromethylbenzyloxy)-propionamide

[0433]

[0434] HPLC Rt=20.73 mins (>90%), HPLC-MS 422.1 [M+H]⁺, 444.1 [M+Na]⁺.

[0435] The required carboxylic acid,2RS-3-Phenyl-2-(4-trifluoromethylbenzyloxy)-propionic acid was preparedfollowing the general method described for compound (12) as a colourlessoil, yield 12.7 mg. HPLC-MS (single main UV peak with Rt=9.59 mins,325.1 [M+H]⁺).

[0436]_(δ)H (CDCl₃ at 298K); 2.80-3.17 (2H, PhCH ₂, m), 3.90-4.14 (1H,OCHCO, m), 4.19-4.37 (1H CH ₂O, d, J=12.57 Hz), 4.45-4.72 (1H, CH ₂°, d,J=12.53 Hz), 6.85-7.46 (9H, ArH, m).

EXAMPLE 26 (2S, 3R)2-Benzyloxy-3-cyclohexyl-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0437]

[0438] HPLC Rt=20.73 mins (>90%), HPLC-MS 360.2 [M+H]⁺, 741.4 [2M+Na]⁺.

[0439] Prepared as detailed for EXAMPLE 1, but using loaded buildingblock-linker construct (9c)

EXAMPLE 27 (2S, 3R)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0440]

[0441] HPLC Rt=19.71 mins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0442] Prepared as detailed for EXAMPLE 3, but using loaded buildingblock-linker construct (9c)

EXAMPLE 28 (2S, 3R)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0443]

[0444] HPLC Rt=19.71 mins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0445] Prepared as detailed for EXAMPLE 4, but using loaded buildingblock-linker construct (9c)

EXAMPLE 29 (2S, 3R)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0446]

[0447] HPLC Rt=17.63 mins (>80%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0448] Prepared as detailed for EXAMPLE 5, but using loaded buildingblock-linker construct (9c)

EXAMPLE 30 (2S, 3R)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0449]

[0450] HPLC Rt=17.93 mins (>95%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0451] Prepared as detailed for EXAMPLE 6, but using loaded buildingblock-linker construct (9c)

EXAMPLE 31 (2S, 3R)2-(4-tert-Butyl-phenylmethanesulfonyl)-4-methyl-pentanoic Acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0452]

[0453] HPLC Rt=21.13 mins (>95%), HPLC-MS 424.2 [M+H]⁺, 869.4 [2M+Na]⁺.

[0454] Prepared as detailed for EXAMPLE 7, but using loaded buildingblock-linker construct (9c)

EXAMPLE 32 (2S, 3R) Morpholine-4-carboxylic Acid2-cyclohexyl-1-(2-methyl-4 oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylEster

[0455]

[0456] HPLC-MS 383.4 [M+H]⁺.

[0457] Prepared as detailed for EXAMPLE 8, but using loaded buildingblock-linker construct (9c)

EXAMPLE 33 (2S, 3R)2-Cyclohexylmethyl-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-4-morpholin-4-yl-4-oxo-butyramide

[0458]

[0459] HPLC-MS 381.4 [M+H]⁺.

[0460] Prepared as detailed for EXAMPLE 9, but using loaded buildingblock-linker construct (9c)

EXAMPLE 34 (2S, 3R) 2-Biphenyl-3-yl-4-methyl-pentanoic acid (2-methyl-1oxo-tetrahydrofuran-3-yl)-amide

[0461]

[0462] HPLC Rt=20.66 mins (>95%), HPLC-MS 366.1 [M+H]⁺, 753.2 [2M+Na]⁺.

[0463] Prepared as detailed for EXAMPLE 10, but using loaded buildingblock-linker construct (9c)

EXAMPLE 35 (2R, 3R)2-Benzyloxy-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-3-phenyl-propionamide

[0464]

[0465] HPLC Rt=18.35 mins (>90%), HPLC-MS 354.2 [M+H]⁺, 376.1 [M+Na]⁺,729.3 [2M+Na]⁺.

[0466] Following the general details from Scheme 1, the required bicyclebuilding block (2R, 3R) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamicacid 9H-fluoren-9-ylmethyl ester (6d) was prepared as follows:

[0467] (1) Preparation of (1R, 1′R)[3-diazo-1-(1-hydroxyethyl)-2-oxopropyl]carbamic Acid9H-fluoren-9-ylmethyl ester.

[0468] A solution of iso-butyl chloroformate (0.751 g, 5.5 mmol) indichloromethane (10 ml) and a solution of 4-methylmorpholine (1.01 g, 10mmol) in dichloromethane (10 ml) were simultaneously added in fourportions to a stirred suspension of Fmoc-D-allo-threonine (1.71 g, 5mmol) in dichloromethane (50 ml) at −15° C. over 10 minutes under anatmosphere of nitrogen. Ethereal diazomethane [generated from diazald(5.1 g, 15 mmol) addition in diethyl ether (82 ml) to sodium hydroxide(5.73 g) in water (10 ml)/ethanol (20 ml) at 60° C.] was then cautiouslyadded and the resulting yellow solution was stirred at room temperaturefor 90 minutes. Acetic acid (2 ml) was cautiously added (untileffervescence had ceased), then the mixture was diluted with tert-butylmethyl ether (50 ml). The ethereal layer was washed with water (3×50ml), dried (Na₂SO₄) and the solvent removed in vacuo to leave (1R, 1′R)[3-diazo-1-(1-hydroxyethyl)-2-oxopropyl]carbamic acid9H-fluoren-9-ylmethyl ester as a yellow solid (2.43 g) which was usedwithout further purification.

[0469] (2) Cyclisation to (2R, 3R)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic Acid 9H-fluoren-9-ylmethylEster.

[0470] A solution of lithium chloride (2.12 g, 50 mmol) in 80% aceticacid in water (75 ml) was added to (1R, 1′R)[3-diazo-1-(1-hydroxyethyl)-2-oxopropyl]carbamic acid9H-fluoren-9-ylmethyl ester (2.43 g, ˜5 mmol). The yellow oilysuspension was stirred for 2 hours whereupon a pale yellow solutionformed accompanied by the evolution of a gas. The solvents were removedin vacuo then the residue was dissolved in ethyl acetate (30 ml), washedwith 10% aqueous sodium carbonate solution (2×30 ml) and saturatedaqueous sodium chloride solution (30 ml). The combined aqueous layerswere extracted with ethyl acetate (2×30 ml) then the combined ethylacetate layers were dried (Na₂SO₄) and the solvents removed in vacuo toleave a yellow gum (1.59 g). The yellow gum was purified bychromatography over silica gel eluting with a gradient ofn-heptane:ethyl acetate 9:1→7:3. Appropriate fractions were combined andthe solvents removed in vacuo to leave (2R, 3R)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic acid 9H-fluoren-9-ylmethylester as a white crystalline solid (796 mg, 47% from starting acid). TLC(single UV spot, Rf=0.27, n-heptane:ethyl acetate 2:1), analytical HPLCpeak Rt=18.04 mins, HPLC-MS (single UV peak with Rt=8.47 mins, 338.1[M+H]⁺, 360.0 [M+Na]⁺, 361.1 [M+H+Na]⁺).

[0471]_(δ)H (CDCl₃ at 298K); 1.51-1.52 (3H, CH ₃CHO, brs), 3.81-3.83(1H, CH₃CHO m), 3.98-4.03 (2H, Fmoc CH ₂, m), 4.14-4.17 (2H, Fmoc H-9and NHCHCO, m), 4.29-4.53 (2H, OCH ₂CO, dd), 5.18-5.19 (1H, NH, bs),7.28-7.32 (2H, ArH, Fmoc H-2 and H-7), 7.34-7.36 (2H, ArH, Fmoc H-3 andH-6), 7.41-7.54 (2H, ArH, Fmoc H-1 and H-8), 7.59-7.80 (2H, ArH, FmocH-4 and H-5).

[0472]_(δ)C (CDCl₃ at 298K); 18.27 (u, CH₃CHO), 46.35 (u, Fmoc C-9),61.91 (u, NHCHCO), 66.46 (d, Fmoc CH₂), 70.03 (d, COCH₂), 76.60 (u,CH₃CHO), 119.26 (u, Fmoc C4 and C-5), 124.21 (u, Fmoc C-1 and C-8),126.35 (u, Fmoc C-2 and C-7), 127.03 (u, Fmoc C-3 and C-6), 140.57 (q,Fmoc C4′ and C-5′), 142.81/142.86 (q, Fmoc C-1′ and C-8′), 155.27(OCON), 211.05 (COCH₂).

[0473] Following the general details from Scheme 2, the required bicyclebuilding (2R, 3R) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6d) was converted to building block-linkerconstruct (8d) as follows:

[0474] (2R, 3R) (2-methyl-4-oxo-tetrahydrofuran-3-yl)-carbamic acid9H-fluoren-9-ylmethyl ester (6d) (354 mg, 1.05 mmol, 1 eq) was dissolvedin a mixture of ethanol (9.6 mL) and water (1.4 mL) containing sodiumacetate.trihydrate (218 mg, 1.6 mmol, 1.5 eq).4-[[(hydrazinocarbonyl)amino] methyl]cyclohexanecarboxylic acid.trifluoroacetate (346 mg, 1.05 mmol, 1 eq, Murphy, A. M. et al, J. Am.Chem. Soc., 114, 3156-3157, 1992) was added and the mixture refluxed for2 hr. Chloroform (100 mL) was added and the organics washed with diluteaqueous hydrochloric acid (0.1 M, 1×100 mL). The acidic layer wasbackwashed with chloroform (3×100 mL) and the combined organics washedwith brine (100 mL), dried (Na₂SO₄) and evaporated under reducedpressure to afford linker construct (8d) as a white solid (620 mg).Analytical HPLC indicated one main peak at R_(t)=17.20 min, HPLC-MS(main UV peak with R_(t)=7.91 min, 535.3 [M+H]⁺. Crude (8d) was useddirectly for construct loading.

[0475] Following the general details from Scheme 2, the requiredbuilding block-linker construct (8d) was attached to the solid phaseproviding loaded building block-linker construct (9d) as follows:

[0476] Building block-linker construct (8d) (1.0 mmole),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro phosphate(HBTU, 379 mg, 1.0 mmole), 1-hydroxybenzotriazole.hydrate and (HOBT, 153mg, 11.0 mmole) were dissolved in dimethylformamide (5 mL) andN-methylmorpholine (NMM, 220 μL, 2.0 mmole) added. After pre-activationfor 5 minutes, free amine gears (250×1.21 mole) were added, followed bydimethylformamide (30 mL) and left overnight. The spent couplingsolution was then added to free amine crowns (25×10 μmole) and leftovernight. Standard washing and analyses indicated loading at >95%.

[0477] Following the general details from Scheme 2, the required loadedbuilding block-linker construct (9d) was elaborated on the solid phaseas follows:

[0478] 2RS-benzyloxy-3-phenylpropionic acid was coupled under standardconditions to loaded building block-linker construct (9d) (followingstandard removal of Fmoc), then cleaved to provide EXAMPLE 35. The crudeexample was analysed (see general techniques). HPLC Rt=18.35 mins(>90%), BPLC-MS 354.2 [M+H]⁺, 376.1 [M+Na]⁺, 729.3 [2M+Na]⁺.

[0479] The following examples (36-47) were prepared as detailed forEXAMPLE 35, coupling with the required reagents to provide the filllength molecule.

EXAMPLE 36 (2R, 3R) 4-Methyl-2-(4-trifluoromethylbenzyloxy)-pentanoicAcid (2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0480]

[0481] HPLC Rt=20.69 mins (>90%), HPLC-MS 388.2 [M+H]⁺, 797.2 [2M+Na]⁺.

[0482] Prepared as detailed for EXAMPLE 12, but using loaded buildingblock-linker construct (9d)

EXAMPLE 37 (2R, 3R)2-Benzyloxy-3-cyclohexyl-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0483]

[0484] HPLC Rt=20.70 mins (>90%), HPLC-MS 360.2 [M+H]⁺, 741.4 [2M+Na]⁺.

[0485] Prepared as detailed for EXAMPLE 1, but using loaded buildingblock-linker construct (9d)

EXAMPLE 38 (2R, 3R)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0486]

[0487] HPLC Rt=19.71 mins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0488] Prepared as detailed for EXAMPLE 3, but using loaded buildingblock-linker construct (9d)

EXAMPLE 39 (2R, 3R)3-Cyclohexyl-2-(furan-2-ylmethylsulfanyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0489]

[0490] HPLC Rt=19.71 miins (>80%), HPLC-MS 366.1 [M+H]⁺, 753.2 [M+Na]⁺.

[0491] Prepared as detailed for EXAMPLE 4, but using loaded buildingblock-linker construct (9d)

EXAMPLE 40 (2R, 3R) 2-(4-tert-Butyl-benzylsulfanyl)-4-methyl-pentanoicacid (2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0492]

[0493] HPLC Rt=23.23 mins (>95%), HPLC-MS 392.2 [M+H]⁺, 805.4 [2M+Na]⁺.

[0494] Prepared as detailed for EXAMPLE 7, but using loaded buildingblock-Linker construct (9d) and no oxidation of the intermediatethioether.

EXAMPLE 41 (2R, 3R)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0495]

[0496] HPLC Rt=17.67 mins (>80%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0497] Prepared as detailed for EXAMPLE 5, but using loaded buildingblock-linker construct (9d)

EXAMPLE 42 (2R, 3R)3-Cyclohexyl-2-(furan-2-ylmethanesulfonyl)-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-propionamide

[0498]

[0499] HPLC Rt=17.93 mins (>95%), HPLC-MS 398.1 [M+H]⁺, 817.1 [M+Na]⁺.

[0500] Prepared as detailed for EXAMPLE 6, but using loaded buildingblock-linker construct (9d)

EXAMPLE 43 (2R, 3R)2-(4-tert-Butyl-phenylmethanesulfonyl)-4-methyl-pentanoic Acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0501]

[0502] HPLC Rt=21.19 mins (>95°/O), HPLC-MS 424.2 [M+H]⁺, 869.4[2M+Na]⁺.

[0503] Prepared as detailed for EXAMPLE 7, but using loaded buildingblock-linker construct (9d)

EXAMPLE 44 (2R, 3R) Morpholine-4-carboxylic acid2-cyclohexyl-1-(2-methyl-4-oxo-tetrahydro-furan-3-ylcarbamoyl)-ethylester

[0504]

[0505] HPLC-MS 383.4 [M+H]⁺.

[0506] Prepared as detailed for EXAMPLE 8, but using loaded buildingblock-linker construct (9d)

EXAMPLE 45 (2R, 3R)2-Cyclohexylmethyl-N-(2-methyl-4-oxo-tetrahydrofuran-3-yl)-4-morpholin-4-yl-4-oxo-butyramide

[0507]

[0508] HPLC-MS 381.4 [M+H]⁺.

[0509] Prepared as detailed for EXAMPLE 9, but using loaded buildingblock-linker construct (9d)

EXAMPLE 46 (2R, 3R) 2-Biphenyl-3-yl-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0510]

[0511] HPLC Rt=20.62 miris (>95%), HPLC-MS 366.1 [M+H]⁺, 753.2 [2M+Na]⁺.

[0512] Prepared as detailed for EXAMPLE 10, but using loaded buildingblock-linker construct (9d)

EXAMPLE 47 (2R, 3S)2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-2oxo-ethyl]-4-methyl-pentanoicAcid (2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0513]

[0514] HPLC Rt=16.04 & 16.54 mins (>80%), HPLC-MS 387.2 [M+H]⁺, 795.4[2M+Na]⁺.

[0515] Prepared as detailed for EXAMPLE 23, but using loaded buildingblock-linker construct (9d)

[0516] Solution Phase Syntheses

[0517] EXAMPLES (48-51) were prepared by standard solution phasechemistries, coupling with the required reagents to provide the fulllength molecule. The core building block (45, SR)4-amino-5-methyl-dihydro-furan-3-one hydrochloride (Compound (31),Schemes 9 and 10) was prepared as follows:

[0518] (1) Preparation of (1S, 1′R)[1-(1-tert-butoxyethyl)-3-diazo-2-oxopropyl]carbamic Acid tert-butylEster

[0519] A solution of iso-butyl chloroformate (0.998 ml, 7.7 mmol) indichloromethane (14 ml) and a solution of 4-methylmorpholine (1.54 ml,14 mmol) in dichloromethane (14 ml) were simultaneously added to astirred suspension of Boc-O-(tert-butyl)-L-threonine (1.93 g, 7 mmol) indichloromethane (70 ml) at −15° C. over 10 minutes under an atmosphereof argon. Ethereal diazomethane [generated from diazald (7.14 g, ˜21mmol) addition in diethyl ether (115 ml) to sodium hydroxide (8.02 g) inwater (11 ml)/ethanol (31 ml) at 60° C.] was then cautiously added andthe resulting yellow solution was stirred at room temperature for 90minutes. Acetic acid (˜2 ml) was cautiously added (until effervescencehad ceased) then the mixture was diluted with tert-butyl methyl ether(50 ml). The ethereal layer was washed with water (3×50 ml), dried(Na₂SO₄) and the solvent removed in vacuo to leave (1S, 1′R)[1-(1-tert-butoxyethyl)-3-diazo-2-oxopropyl]carbamic acid tert-butylester as a yellow solid (2.81 g) which was used without furtherpurification.

[0520] (2) Cyclisation to (2R, 3S)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic Acid tert-butyl Ester

[0521] A solution of lithium chloride (2.97 g, 70 mmol) in 80% aceticacid in water (105 ml) was added to (1S,1′R)[1-(1-tert-butoxyethyl)-3-diazo-2-oxopropyl]carbamic acid tert-butylester (2.81 g, ˜7 mmol). The yellow oily suspension was stirred for 2hours whereupon a pale yellow solution formed accompanied by theevolution of a gas. The solvents were removed in vacuo then the residuewas dissolved in ethyl acetate (42 ml), washed with 10% aqueous sodiumcarbonate solution (2×42 ml) and saturated aqueous sodium chloridesolution (42 ml). The combined aqueous layers were extracted with ethylacetate (2×42 ml) then the combined ethyl acetate layers were dried(Na₂SO₄) and the solvents removed in vacuo to leave a yellow gum (1.1g). The yellow gum was purified by chromatography over silica geleluting with n-heptane:ethyl acetate 3:1 Appropriate fractions werecombined and the solvents removed in vacuo to leave (2R, 3S)(2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamic acid tert-butyl ester as awhite crystalline solid (393 mg, 26% from starting acid). TLC (single UVspot, Rf=0.31, n-heptane:ethyl acetate 2:1), HPLC-MS (single UV peakwith Rt=6.448 mins, 238.1 [M+Na]⁺, 453.3 [2M+Na]⁺).

[0522]_(δ)H (CDCl₅ at 298K); 1.47 (9H, G(CH ₃)₃, s), 1.53 (3H, CH ₃CHO,d, J=6.02 Hz), 3.68-3.81 (1H, CH₃CHO, m), 3.90-4.05 (2H, COCH ₂O andNHCHCO, m), 4.20-4.31 (1H, OCH ₂CO, d, J=17.36 Hz), 4.84-4.97 (1H, NH,brs).

[0523]_(δ)C (CDCl₃ at 298K); 19.50 (u, CH₃CHO), 28.61 (u, C(CH₃)₃),62.91 (u, Cα), 71.23 (d, COCH₂), 78.06 (u, CH₃ CHO), 81.03 (q, C(CH₃)₃),155.73 (q, OCON), 212.85(q, COCH₂).

[0524] (3) Preparation of (45, 5R) 4-amino-5-methyl-dihydrofuran-3-oneHydrochloride.

[0525] HCl in dioxane (4.0M, 9 ml) was added drop-wise with stirringover 1 minute to (2R, 3S) (2-methyl-4-oxo-tetrahydrofuran-3-yl)carbamicacid tert-butyl ester (0.372 g, 1.73 mmol). The mixture was stirred for20 minutes then the solvents evaporated in vacuo to leave a residuewhich was azeotroped with toluene (2×20 ml) to leave (4S, 5R)4amino-5-methyl-dihydro-furan-3-one hydrochloride as a white solid (266mg, 100%). HPLC-MS, Rt=0.419 mins, 116.1 [M+H]⁺).

EXAMPLE 48 (2R, 3S) 24tert-Butylbenzyloxy)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0526]

[0527] HPLC Rt=21.429 mins, HPLC-MS 376.2 [M+H]⁺, 773.4 [2M+Na]⁺.

[0528] The required ether carboxylic acid building block2RS-(4-tert-butyl-benzyloxy)-4-methyl-pentanoic acid (compound (33),Scheme 10) was prepared from (S)-2-hydroxyisocaproic acid (32) asfollows:

[0529] (1) Preparation of 2RS-(4-tert-butylbenzyloxy)-4-methyl-pentanoicAcid (Compound (33), Scheme 10)

[0530] A solution of (S)-2-hydroxyisocaproic acid (compound (32) 0.66 g,5 mmol) in dichloromethane (10 ml) was added to a suspension of sodiumhydride (0.44 g of 60% dispersion in oil, 11 mmol) in dimethylformamide(20 ml) at 0° C. The mixture was stirred at 0° C. for 0.5 hours then asolution of 4-tert-butylbenzyl bromide (1.42 g, 6.25 mmol) indichloromethane (5 ml) was added drop-wise. The suspension was stirredfor 2 hours then 1M hydrochloric acid (40 ml) was added and the productextracted into dichloromethane (100 ml) then washed with saturatedaqueous sodium chloride solution (2×50 ml), dried (Na₂SO₄) and thesolvent removed in vacuo to leave a residue. The residue was purified bychromatography over silica gel eluting with methanol:dichloromethane1:100. Appropriate fractions were combined and the solvents removed invacuo to leave 2RS-(4-tert-butylbenzyloxy)-4-methyl-pentanoic acid as ayellow oil (79 mg, 6%). Analytical HPLC peak Rt=21.310 mins, HPLC-MS(single peak with Rt=10.424 mins, 301.1 [M+Na]⁺, 579.2 [2M+Na]⁺).

[0531]_(δ)H (CDCl₃ at 298K); 0.71 and 0.88 (6H, CH₂CH(CH ₃)₂, both d,J=6.6 Hz), 1.23 (9H, C(CH ₃)₃, s), 1.42-1.54 (1HCH ₂CH(CH₃)₂, m),1.63-1.74 (1HCH ₂CH(CH₃)₂, m), 1.75-1.87 (1H, CH₂CH(CH₃)₂, m), 3.88-3.97(1H, OCHCOOH, m), 4.28-4.35 (1H, ArCH ₂O, d, J=11.26 Hz), 4.55-4.67 (1H,ArCH ₂O, d, J=11.25 Hz), 7.20-7.33 (4H, ArH, m).

[0532]_(δ)C (CDCl₃ at 298K); 23.61 (u, CH₂ CH(CH₃)₂), 24.83 (u,CH₂CH(CH₃)₂), 31.51 (u, C(CH₃)₃), 42.14 (d, CH ₂CH(CH₃)₂), 72.68 (d,ArCH₂O), 76.54 (u, OCHCOOH), 125.75/125.84 (u, aromatic CH),128.29/128.48 (u, aromatic CH), 134.71 (q, Ar C), 151.36 (q, Ar C),178.41 (q, CO).

[0533] Carboxylic acid building (33) and (4S, 5R)4-amino-5-methyl-dihydro-furan-3-one hydrochloride (31) were coupled asfollows:

[0534] iso-Butyl chloroformate (20.09 mg, 0.1471 mmol) indichloromethane (2 ml) and N-methyl morpholine (29.75 mg, 0.294 mmol) indichloromethane (2 ml) were simultaneously added were simultaneouslyadded to a stirred solution of (31) (37.22 mg, 0.1337 mmol) indichloromethane (2 ml) at −15° C. under argon over 5 minutes. Themixture was stirred at −15° C. for 15 minutes. A freshly preparedsolution of (33) (20.27 mg, 0.1337 mmol) in dichloromethane (2 ml) and4-methylmorpholine (13.52 mg, 0.1337 mmol) was then added dropwise. Themixture was then stirred at ambient temperature for 16 hours. Thesolvent was removed in vacuo to give a residue which was purified oversilica gel eluting with a gradient of n-heptane:ethyl acetate 3:1 to2:1. Desired fractions were combined and reduced in vacuo to leave awhite foam (37.10 mg, 73.9% from starting acid). TLC (single UW spot,Rf=0.85, 10% MeOH in DCM), analytical HPLC single peak with Rt=21.429mins, HPLC-MS (single UV peak with Rt=10.829 mins, 376.2 [M+H]⁺, 773.4[2M+Na]⁺.

[0535]_(δ)H (CDCl₃ at 298K); 0.75-0.90 (6H, (CH ₃)₂CH₂, m), 1.25 (9H,C(CH ₃)₃, s), 1.34-1.41 (3H, OCHCH ₃, d, J=6.03 Hz), 1.45-1.63 (2H, CH₂CH(CH₃)₂, m), 1.71-1.87 (1H, CH₂CH(CH₃)₂, m), 3.71-3.80 (1H, OCHCH₃,m), 3.83-4.04 (3H, OCHCO, NHCHCO and COCH ₂O, m), 4.12-4.21 (1H, COCH₂O, m), 4.47 (2H, ArCH ₂, s), 6.77 (1H, NH, s), 7.18-7.36 (4H, ArH, m).

EXAMPLE 49 (2R,3S)N-(2-Methyl-4-oxo-tetrahydrofuran-3-yl)-2-(naphthalen-1-ylmethoxy)-3-phenyl-propionamide

[0536]

[0537] Analytical HPLC Rt=20.50 mins (major isomer) and 20.64 mins(minor isomer) and HPLC-MS (single major UV peak with Rt=9.746 mins,404.2 [M+H]⁺, 426.2 [M+Na]⁺, 829.4 [2M+Na]⁺).

[0538] The required ether carboxylic acid building block2RS-(Naphthalen-1-ylmethoxy)-3-phenylpropionic acid was prepared asfollows:

[0539] Sodium hydride (265 mg of 60% dispersion in oil, 6.6 mmol) wasadded in two portions to a stirred mixture of(S)-2-hydroxy-3-phenylpropionic acid (0.5 g, 3.0 mmol),dimethylformamide (5 ml) and dichloromethane (5 ml) at 0° C. over 5minutes. The mixture was stirred at 0° C. for 5 minutes then at ambienttemperature for 30 minutes. 1-(Chloromethyl)naphthalene (95%, 0.57 ml,3.6 mmol) was added then the mixture stirred for 5 hours before addingpotassium iodide (50 mg, 0.3 mmol). The mixture was stirred for 20 hoursthen heated at 55° C. for 1 hour then allowed to cool to ambienttemperature and poured into water (15 ml). A saturated aqueous sodiumchloride solution (5 ml) was added then the mixture was extracted withdichloromethane (5 ml then 10 ml) that was discarded. The aqueous layerwas acidified using 1M hydrochloric acid (10 ml) then extracted withdichloromethane (2×10 ml). The dichloromethane layer was dried (MgSO₄)and the solvent removed in vacuo. The residue (0.48 g) was dissolved indimethylformamide (6 ml) then cooled to 0° C. before adding sodiumhydride (150 mg of 60% dispersion in oil, 3.75 mmol). The mixture wasstirred for 30 minutes then polymer bound isocyanate (300 mg, 2 mmolNg⁻¹) added. The mixture was stirred for 2 hours at ambient temperaturethen poured into water (15 ml). 1M Hydrochloric acid (10 ml) was addedthen the product extracted into dichloromethane (2×10 ml), dried (MgSO₄)and the solvent removed in vacuo. The residue was dissolved in aqueoussodium hydroxide solution (1M, 10 ml) then extracted withdichloromethane (2×10 ml) that was discarded. Crushed ice was added tothe aqueous layer followed by 1M hydrochloric acid (12 ml). The productwas extracted into dichloromethane (2×10 ml) then dried (Na₂SO₄) and thesolvent removed in vacuo. The residue was purified by flashchromatography over silica gel eluting with a gradient ofmethanol:dichloromethane 0:1→1:20. Appropriate fractions were combinedand the solvents removed in vacuo to leave2RS-(Naphthalen-1-ylmethoxy)-3-phenylpropionic acid (40 mg) as acolourless oil. HPLC-MS (single main UV peak with Rt=9.22 mins, 307.2[M+H]⁺, 329.2 [M+Na⁺).

[0540]_(δ)H (CDCl₃ at 298K); 2.82-3.18 (2H, PhCH ₂, m), 4.00-4.25 (1H,OCHCO, m), 4.63-4.81 (1H, CH ₂O, d, J=11.55 Hz), 4.92-5.10 (1H CH ₂O, d,J=11.52 Hz), 6.90-7.86 (12H, ArH, m), 8.78 (1H, OH, brs).

[0541] 2RS-(Naphthalen-1-ylmethoxy)-3-phenylpropionic acid and (4S, SR)4-amino-5-methyl-dihydro-furan-3-one hydrochloride (31) were coupled asfollows:

[0542] Solutions of isobutyl chloroformate (18.6 μl, 0.14 mmol) indichloromethane (0.67 ml) and 4-methylmorpholine (31.6 μl, 0.29 mmol) indichloromethane (0.67 ml) were added simultaneously at −15° C. over 10minutes drop-wise to a stirred solution of2RS-Naphthalen-1-ylmethoxy)-3-phenylpropionic acid (40 mg, 0.13 mmol) indichloromethane (0.5 ml) under argon. The solution was stirred for 15minutes then (45, SR) 4-amino-5-methyldihydrofuran-3-one hydrochloride(20 mg, 0.13 mmol) followed by a solution of 4-methylmorpholine (14.4μl, 0.13 mmol) in dichloromethane (0.3 ml) were added. The mixture wasallowed to warm to 0° C. over 3 hours, stirred at ambient temperaturefor 20 hours then the solvents were removed in vacuo. The residue waspurified by flash chromatography over silica gel eluting with a gradientof ethyl acetate:heptane 1:3→7:13. Appropriate fractions were combinedand the solvents removed in vacuo to leave (2R,3S)N-(2-Methyl-4-oxo-tetrahydrofuran-3-yl)-2-(naphthalen-1-ylmethoxy)-3-phenyl-propionamideas a white solid (28.5 mg, 3.5:1 mixture of diastereoisomers, 70%). TLC(two spots, Rf=0.45 and 0.42, ethyl acetate:heptane 7:13).

[0543]_(δ)H (CDCl₃ at 298K); Major isomer 1.4 (3H, C{overscore (H)}₃CH,d, J=6.0 Hz), 2.90 (1H, PhC{overscore (H)}₂, dd, J=14.1, 8.0 Hz), 3.15(1H, PhC{overscore (H)}₂, dd, J=14.1, 3.6 Hz), 3.32 and 3.51 (both 1H,CH₃CHCHN, m), 3.85 (1H, OCH ₂CO, d, J=17.1 Hz), 4.08 (1H, OCH ₂CO, d,J=17.1 Hz), 4.19 (1H, OCHCO, m), 4.6 (1H, OCH ₂Naphthyl, d, J=11.8 Hz),4.90 (1H, OCH ₂Naphthyl, d, J=11.8 Hz), 6.55 (1H, NH, d, J=6 Hz),7.18-7.93 (12H, aromatic); Minor isomer 1.50 (3H, CH ₃CH, d, J=6.0 Hz),2.94 (1H, PhCH ₂, m), 3.15 (1H, PhCH ₂, m), 3.52 and 3.89 (both 1H,CH₃CHCHN, m), 4.02-4.22 (3H, OCH ₂CO and OCHCO, m), 4.6 (2H, OCH₂Naphthyl, s), 6.74 (1H, NH, d, J=6 Hz), 7.18-7.93 (12H, aromatic).

EXAMPLE 50 (2R, 3S) 2-(Biphenyl-4-ylmethoxy)-4-methyl-pentanoic Acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide

[0544]

[0545] HPLC Rt=20.178 mins (>95%), HPLC-MS 396.2 [M+H]⁺, 813.4 [2M+Na]⁺.

[0546] The required ether carboxylic acid building block 2RS2-(Biphenyl-4-ylmethoxy)₄ methyl-pentanoic acid was prepared as follows:

[0547] (1) Preparation of 2RS-(Biphenyl-4-ylmethoxy)4-methyl-pentanoicAcid Methyl Ester

[0548] Sodium hydride (98.4 mg of 60% dispersion in oil, 2.46 mmol) wasadded in two portions to a stirred solution of2S-hydroxy-4-methyl-pentanoic acid methyl ester (300 mg, 2.05 mmol) intetrahydrofuran (2.5 ml) at 0° C. under an atmosphere of nitrogen over 5minutes. The mixture was stirred at ambient temperature for 10 minutesthen a solution of 4-phenylbenzylchloride (830 mg, 4.1 mmol) followed bypotassium iodide (25 mg) was added. The mixture was stirred for 20 hoursthen saturated aqueous ammonium chloride solution (40 ml) was added. Theproduct was extracted into ethyl acetate (3×30 ml) then the combinedethyl acetate layers were washed with aqueous saturated sodium chloridesolution, dried (Na₂SO₄) and the solvents removed in vacuo. The residue(1.2 g) was purified by flash chromatography over silica gel elutingwith ethyl acetate:heptane 1:20→1:9. Appropriate fractions were combinedand the solvents removed in vacuo to leave3-cyclohexyl-2-(furan-3-ylmethoxy)-propionic acid methyl ester (110 mg,17.1%) as a colourless oil. TLC (single spot, Rf=0.2, heptane:ethylacetate 9:1), HPLC-MS main UV peak with 313.1 [M+H]⁺.

[0549] (2) Preparation of 2RS-(Biphenyl-4-ylmethoxy)-4-methyl-pentanoicAcid.

[0550] A solution of lithium hydroxide monohydrate (0.0443 g, 1.056mmol) in water (3 ml) was added drop-wise over 1 minute to a solution of2-(biphenyl]-ylmethoxy)-4-methyl-pentanoic acid methyl ester (0.11 g,0.3521 mmol) in tetrahydrofuran methanol 2:1 (15 ml) at 0° C. Themixture was stirred at ambient temperature for 16 hours then poured intosaturated aqueous sodium chloride solution (10 ml). The aqueous layerwas extracted with diethyl ether (2×10 ml) which was discarded. Theaqueous layer was acidified with 1M hydrochloric acid to pH (1˜2), thenthe product was extracted into dichloromethane (3×10 ml). The combineddichloromethane layers were washed with water (2×10 ml), saturatedaqueous sodium chloride solution (2×10 ml) then dried (Na₂SO₄) and thesolvents removed in vacuo to leave2-(biphenyl-4-ylmethoxy)-4-methyl-pentanoic acid as a colourless oil(0.048 g, 45.69%). TLC (single UV spot, Rf=0.2, methanol:dichloromethane0.5:9.5), HPLC-MS (297.1 [M−1)⁺, 321.1 [M+Na]⁺, 619.3 [2M+Na]⁺).

[0551]_(δ)H (CDCl₃ at 298K); 0.71-0.94 (6H, (CH ₃)₂CH, two d, J1a=6.57,J1b=6.66 Hz, J2=29.87 Hz), 1.19 (1H, OH, bs,), 1.45-1.76 (2H, CH₂CH(CH₃)₂, m), 1.77-1.92 (1H, CH₂CH(CH₃)₂, m), 2.93-4.05 (1H, CHCOOH,m), 4.37-4.48 (1H, CH ₂Ar, d, J=11.43 Hz), 4.64-4.75 (1H, CH ₂Ar, d,J=11.42 Hz), 7.23-7.60 (9H, ArH, m).

[0552]_(δ)C (CDCl₃ at 298K); 23.17 (U, (CH₃)₂ CH), 24.81/26.13 (u,(CH₃)₂CH), 43.30 (d, CH₃)₂CHCH₂), 74.06 (d, ArCH₂), 78.02 (u, OCHCOOH),128.77/128.91/129.03.43 (u, Ar), 130.31/130.44 (u, Ar), 137.71 (q, Ar),142.42/142.71 (q, Ar), 179.79 (q, OCO).

[0553] 2RS-2-(Biphenyl-4-ylmethoxy)-4-methyl-pentanoic acid and (4S, 5R)4-amino-5-methyl-dihydro-furan-3-one hydrochloride (31) were coupled asfollows:

[0554] iso-Butyl chloroformate (15.61 mg, 0.1143 mmol) indichloromethane (2 ml) and N-methyl morpholine (23.12 mg, 0.2286 mmol)in dichloromethane (2 ml) were simultaneously added were simultaneouslyadded to a stirred solution of2RS-2-(biphenylylmethoxy)-4-methyl-pentanoic acid (31 mg, 0.104 mmol) indichloromethane (2 ml) at −15° C. under argon over 5 minutes. Themixture was stirred at −15 IC for 15 minutes. A freshly preparedsolution of (31) (15.75 mg, 0.104 mmol) in dichloromethane (2 ml) and4-methylmorpholine (11.56 mg, 0.1143 mmol) was then added dropwise. Themixture was then stirred at ambient temperature for 16 hours. Thesolvent was removed in vacuo to give a residue which was purified oversilica gel eluting with a gradient of n-heptane: ethyl acetate 3:1.Desired fractions were combined and reduced in vacuo to leave (2R, 3S)2-(Biphenyl-4-ylmethoxy)-4-methyl-pentanoic acid(2-methyl-4-oxo-tetrahydrofuran-3-yl)-amide as an off-white gum (34 mg,82.7% from starting acid). TLC (single UV spot, Rf=0.85, 10% MeOH inDCM).

[0555]_(δ)H (CDCl₃ at 298K); 0.72-0.93 (6H, (CH ₃)₂CH₂, m), 1.39 (3H,OCHCH ₃, d, J=6.04 Hz), 1.45-1.69 (2H, CH ₂CH(CH₃)₂, m), 1.73-1.89 (1H,CH₂CH(CH₃)₂, m), 3.70-3.81 (1H, OCHCH₃, m), 3.84-4.08 (3H, OCHCONH, COCH₂O and NHCHCOCH₂, m), 4.10-4.20 (1H, COCH ₂O, d, J=17.35 Hz), 4.40-4.67(2H, ArCH ₂O, m), 6.71 (1H, NH, bs), 7.60-7.24 (9H, ArH, m).

[0556]_(δ)C (CDCl₃ at 298K); 17.19/17.27 (u, CH₃, furanone),19.70/19.89/19.94 (u, CH₂ CH(CH₃)₂), 21.38/21.43/22.74/22.79 (u,CH₂CH(CH₃)₂), 40.42/40.59/40.80 (d, OCHCH₂CH(CH₃)₂), 59.29 (u,NHCHCOCH₂), 69.17/69.24 (d, ArCH₂O), 70.93/71.05/71.28 (d, COCH₂), 74.68(u, Cβ), 77.11/77.22 (u, OCHCONH),125.22/125.51/125.57/125.60/125.63/126.68/126.97/127.18 (u, Ar CH),133.81/133.98/134.11 (q, Ar C), 138.69/138.75/139.40/139.43 (q, Ar C,171.99/172.23 (q, CON), 209.36/209.98/210.21/172.23 (q, COCH₂).

EXAMPLE 51 (2R, 3S)3-Cyclohexyl-2-(furan-3-ylmethoxy)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamide

[0557]

[0558] HPLC Rt=17.858 mins (>90%), HPLC-MS 350.2 [M+H]⁺, 372.2 [M+Na]⁺,721.3 [2M+Na]⁺.

[0559] The required ether carboxylic acid building block 2RS3-Cyclohexyl-2-(furan-3-ylmethoxy)-propionic acid was prepared asfollows:

[0560] (1) Preparation of 3-cyclohexyl-2-(furan-3-ylmethoxy)-propionicAcid Methyl Ester.

[0561] Sodium hydride (204 mg of 60% dispersion in oil, 5.1 mmol) wasadded in two portions to a stirred solution of3-cyclohexyl-2S-hydroxypropionic acid methyl ester (18) (794 mg, 4.27mmol) in tetrahydrofuran (5 ml) at 0° C. under an atmosphere of nitrogenover 5 minutes. The mixture was stirred at ambient temperature for 10minutes then a solution of 3-bromomethylfuran (2.75 g, 17.08 mmol) intetrahydrofuran (1 ml) was added. The mixture was stirred for 20 hoursthen saturated aqueous ammonium chloride solution (40 ml) was added. Theproduct was extracted into ethyl acetate (3×30 ml) then the combinedethyl acetate layers were washed with aqueous saturated sodium chloridesolution, dried (Na₂SO₄) and the solvents removed in vacuo. The residue(2.0 g) was purified by flash chromatography over silica gel elutingwith ethyl acetate:heptane 1:4. Appropriate fractions were combined andthe solvents removed in vacuo to leave3-cyclohexyl-2-(furan-3-ylmethoxy)-propionic acid methyl ester (64 mg,6%) as a colourless oil. TLC (single spot, Rf=0.2, heptane: ethylacetate 9:1), HPLC-MS (main UV peak ¹with Rt=10.701 mins, 289.1 [M+Na]⁺,555.3 [2M+Na]⁺).

[0562] (2) Preparation of 3-cyclohexyl-2-(furan-3-ylmethoxy)-propionicAcid.

[0563] A solution of lithium hydroxide monohydrate (0.03025 g, 0.721mmol) in water (3 ml) was added drop-wise over 1 minute to a solution of3-cyclohexyl-2-(furan-3-ylmethoxy)-propionic acid methyl ester (0.064 g,0.240 mmol) in tetrahydrofuran: methanol 2:1 (15 ml) at 0° C. Themixture was stirred at ambient temperature for 16 hours then poured intosaturated aqueous sodium chloride solution (10 ml). The aqueous layerwas extracted with diethyl ether (2×10 ml) which was discarded. Theaqueous layer was acidified with 1M hydrochloric acid to pH (1˜2), thenthe product was extracted into dichloromethane (3×10 ml). The combineddichloromethane layers were washed with water (2×10 ml), saturatedaqueous sodium chloride solution (2×10 ml) then dried (Na₂SO₄) and thesolvents removed in vacuo to leave Preparation of3-cyclohexyl-2-(fiuran-3-ylmethoxy)-propionic acid as a light yellow oil(0.012 g, 19.80%). TLC (single UV spot, Rf=0.35,methanol:dichloromethane 1:9), HPLC-MS (252.1 [M]⁺, 275.2 [M+Na]⁺, 527.2[2M+Na]⁺).

[0564] 2RS 3-Cyclohexyl-2-(furan-3-ylmethoxy)-propionic acid and (4S,SR) 4-amino-5-methyl-dihydro-furan-3-one hydrochloride (31) were coupledas follows:

[0565] iso-Butyl chloroformate (7.15 mg, 0.0523 mmol) in dichloromethane(2 ml) and N-methyl morpholine (10.58 mg, 0.1046 mmol) indichloromethane (2 ml) were simultaneously added to a stirred solutionof 2RS 3-Cyclohexyl-2-(furan-3-ylmethoxy)-propionic acid (12.00 mg,0.048 mmol) in dichloromethane (2 ml) at −15° C. under argon over 5minutes. The mixture was stirred at −15° C. for 15 minutes. A freshlyprepared solution of (31) (7.21 mg, 0.048 mmol) in dichloromethane (2ml) and 4-methylmorpholine (5.29 mg, 0.0523 mmol) was then addeddropwise. The mixture was then stirred at ambient temperature for 16hours. The solvent was removed in vacuo to give a residue which waspurified over silica gel eluting with a gradient of n-heptane:ethylacetate 3:1. Desired fractions were combined and reduced in vacuo toleave (2R, 3S)3-Cyclohexyl-2-(furan-3-ylmethoxy)-N-(2-methyl-4-oxo-tetrahydro-furan-3-yl)-propionamideas an off-white gum (6 mg, 35.77% from starting acid). TLC (single spot,Rf=0.8, methanol: dichloromethane 1:9), analytical

[0566]_(δ)H (CDCl₃ at 298K); 0.70-0.95 (2H, CH₂ (cyclohexyl), m),1.02-1.25 (2H, CH₂ (cyclohexyl), m), 1.38 (3H, OCHCH ₃, d, J=6.12 Hz),1.45-1.70 (8H, CH, CH₂ (cyclohexyl), m), 3.70-3.79 (1H, OCHCH₃, m),3.804.08 (3H, OCHCONH, COCH ₂O and NHCHCOCH₂, m), 4.144.23 (1H, COCH ₂O,d, J=17.20 Hz), 4.30-4.46 (2H, 3-Furan-CH ₂O, m), 6.35 (1H, 2-Furan-CH,s), 6.76 (1H, NH, bs), 7.37 (2H, 4 and 5-Furan-CH, d, J=1.67 Hz).

EXAMPLE A Assays for Cysteine Protease Activity

[0567] The compounds of this invention may be tested in one of a numberof literature based biochemical assays that are designed to elucidatethe characteristics of compound inhibition. The data from these types ofassays enables compound potency and the rates of reaction to be measuredand quantified. This information, either alone or in combination withother information, would allow the amount of compound required toproduce a given pharmacological effect to be determined.

[0568] General Materials and Methods

[0569] Unless otherwise stated, all general chemicals and biochemicalswere purchased from either the Sigma Chemical Company, Poole, Dorset,U.K. or from Fisher Scientific UK, Loughborough, Leicestershire, U.K.Absorbance assays were carried out in flat-bottomed 96-well plates(Spectra; Greiner Bio-One Ltd., Stonehouse, Gloucestershire, U.K.) usinga SpectraMax PLUS384 plate reader (Molecular Devices, Crawley, U.K.).Fluorescence high throughput assays were carried out in either 384-wellmicrotitre plates (Corning Costar 3705 plates, Fisher Scientific) or96-well ‘U’ bottomed Microfluor W1 microtitre plates (Thermo Labsystems,Ashford, Middlesex, U.K.). Fluorescence assays were monitored using aSpectraMax Gemini fluorescence plate reader (Molecular Devices). Forsubstrates employing either a 7-amino-4-methylcoumarin (AMC) or a7-amino-4-trifluoromethylcoumarin (AFC) fluorophore, assays weremonitored at an excitation wavelength of 365 nm and an emissionwavelength of 450 nm and the fluorescence plate reader calibrated withAMC. For substrates employing a 3-amino-benzoyl (Abz) fluorophore,assays were monitored at an excitation wavelength of 310 nm and anemission wavelength of 445 nm; the fluorescence plate reader calibratedwith 3-amino-benzamide (Fluka). Unless otherwise indicated, all thepeptidase substrates were purchased from Bachem UK, St. Helens,Merseyside, UK. Substrates utilizing fluorescence resonance energytransfer methodology (i.e. FRET-based substrates) were synthesized atIncenta Limited using published methods (Atherton & Sheppard, SolidPhase Peptide Synthesis, IRL Press, Oxford, U.K., 1989) and employed Abz(2-aminobenzoyl) as the fluorescence donor and 3-nitro-tyrosine[Tyr(NO₂)] as the fluorescence quencher (Meldal, M. and Breddam, K.,Anal. Biochem., 195, 141-147, 1991). Hydroxyethylpiperazineethanesulfonate (HEPES), tris-hydroxylmethyl aminomethane (tris) base,bis-tris-propane and all the biological detergents (e.g. CHAPS,zwittergents, etc.) were purchased from CN Biosciences UK, Beeston,Nottinghamshire, U.K. Glycerol was purchased from Amersham PharmaciaBiotech, Little Chalfont, Buclinghamshire, U.K. Stock solutions ofsubstrate or inhibitor were made up to 10 mM in 100% dimethylsulfoxide(DMSO) (Rathburns, Glasgow, U.K.) and diluted as appropriately required.In all cases the DMSO concentration in the assays was maintained at lessthan 1% (vol./vol.).

[0570] Assay protocols were based on literature precedent (Table 1;Barrett, A. J., Rawlings, N. D. and Woessner, J. F., 1998, Handbook ofProteolytic Enzymes, Academic Press, London and references therein) andmodified as required to suit local assay protocols. Enzyme was added asrequired to initiate the reaction and the activity, as judged by thechange in fluorescence upon conversion of substrate to product, wasmonitored over time. All assays were carried out at 25±1° C. TABLE 1 Theenzyme assays described herein were carried out according to literatureprecedents. Enzyme Buffer Substrate Reference Cathepsin B IZ-Phe-Arg-AMC a, b Cathepsin H II Bz-Phe-Val-Arg-AMC a, b Cathepsin L IAc-Phe-Arg-AMC b, c Cathepsin S I Boc-Val-Leu-Lys-AMC c, d Caspase 1 IIIAc-Leu-Glu-His-Asp-AMC e Caspase 2 III Z-Val-Asp-Val-Ala-Asp-AFC fCaspase 3 III Ac-Asp-Glu-Val-Asp-AMC g, h Caspase 4 IIISuc-Tyr-Val-Ala-Asp-AMC f Caspase 5 III Ac-Leu-Glu-His-Asp-AMC Caspase 6III Ac-Val-Glu-Ile-Asp-AMC i, j, k Caspase 7 III Ac-Asp-Glu-Val-Asp-AMCCaspase 8 III Ac-Ile-Glu-Thr-Asp-AMC l Caspase 9 IIIAc-Leu-Glu-His-Asp-AMC Caspase 10 III Ac-Ile-Glu-Thr-Asp-AMC CruzipainIV D-Val-Leu-Lys-AMC m, n CPB2.8ΔCTE XI Pro-Phe-Arg-AMC q S. Aureus IAbz-Ile-Ala-Ala-Pro- o Extracellular Tyr(NO₂)-Glu-NH₂ cysteine peptidaseClostripain Z-Gly-Gly-Arg-AMC p FMDV LP V Abz-Arg-Lys-Leu-Lys-Gly- rAla-Gly-Ser-Tyr(NO₂)-Glu- NH₂ Trypsin VI Z-Gly-Gly-Arg-AMC s Calpain μVII Abz-Ala-Asn-Leu-Gly-Arg-Pro- t Ala-Leu-Tyr(NO₂)-Asp-NH₂ Calpain mVIII Abz-Lys-Leu-Cys(Bzl)-Phe-Ser- t Lys-Gln-Tyr(NO₂)-Asp-NH₂ CathepsinK IX Z-Phe-Arg-AMC u Cathepsin X X v, w

[0571]Trypanosoma cruzi Cruzipain Peptidase Activity Assays

[0572] Wild-type cruzipain, derived from Trypanosoma cruzi Dm28epimastigotes, was obtained from Dr. Julio Scharfstein (Instituto deBiofisica Carlos Chagas Filho, Universidade Federal do R10 de Janeiro,R10 de Janeiro, Brazil). Activity assays were carried out in 100 mMsodium phosphate, pH 6.75 containing 1 mM EDTA and 10 mM L-cysteineusing 2.5 nM enzyme. Ac-Phe-Arg-AMC (K_(M) ^(app)≈12 μM) andD-Val-Leu-Lys-AMC (K_(M) ^(app)≈4 μM) were used as the substrates.Routinely, Ac-FR-AMC was used at a concentration equivalent to K_(M)^(app) and {overscore (D)}-Val-Leu-Lys-AMC was used at a concentrationof 25 μM. The rate of conversion of substrate to product was derivedfrom the slope of the increase in fluorescence monitored continuouslyover time.

[0573]Leishmania mexicana Cysteine Protease B (CPB) Peptidase ActivityAssays

[0574] Wild-type recombinant CPB without the C-terminal extention (i.e.CPB2.8ΔCTE; Sanderson, S. J., et. al., Biochem. J, 347, 383-388, 2000)was obtained from Dr. Jeremy Mottram (Wellcome Centre for MolecularParasitology, The Anderson College, University of Glasgow, Glasgow,U.K.). Activity assays were carried out in 100 mM sodium acetate; pH 5.5containing 1 mM EDTA; 200 nM NaCl and 10 mM DTT (Alves, L. C., et. al.,Mol. Biochem. Parasitol, 116, 1-9, 2001) using 0.25 nM enzyme.Pro-Phe-Arg-AMC (K_(M) ^(app)=3811M) was used as the substrate at aconcentration equivalent to K_(M) ^(app). The rate of conversion ofsubstrate to product was derived from the slope of the increase influorescence monitored continuously over time.

[0575] Cathepsin Peptidase Activity Assays

[0576] Bovine cathepsin S, human cathepsin L, human cathepsin H andhuman cathepsin B were obtained from CN Biosciences. Recombinant humancathepsin S, human cathepsin K and human cathepsin X were obtained fromDr. Boris Turk (Josef Stefan Institute, Ljubljana, Slovenia). Unlessotherwise stated, all peptidase activity assays were carried out in 10mM bis-tris-propane (BTP), pH 6.5 containing 1 mM EDTA, 5 mM2-mercaptoethanol and 1 mM CaCl₂. Human cathepsin H activity assays werecarried out in 10 mM BTP pH 6.5, 142 mM NaCl₂, 1 mM CaCl₂, 1 mM EDTA, 1mM DTT, 0.035 mM Zwittergent 3-16. Human cathepsin K assays were carriedout in 100 mM sodium acetate; pH 5.5 containing 20 mM L-cysteine and 1mM EDTA (Bossard, M. J., et. al., J. Biol. Chem., 2, 12517-12524, 1996).Human cathepsin X assays were carried out in 100 mM sodium acetate; pH5.5 containing 20 mM L-cysteine; 0.05% (w/v) Brij 35 and 1 mM EDTA(Santamaria, I., et. al., J. Biol. Chem., 2,73, 16816-16823, 1998;Klemencic, J, et al., Eur. J. Biochem., 267, 5404-5412, 2000). The finalenzyme concentrations used in the assays were 0.5 nM bovine cathepsin S,1 nM cathepsin L, 0.1 nM cathepsin B, 0.25 nM Cathepsin K; 1 nMcathepsin X and 10 nM cathepsin H. For the inhibition assays, thesubstrates used for cathepsin S, cathepsin L, cathepsin B, cathepsin Kand cathepsin H were boc-Val-Leu-Lys-AMC (K_(M) ^(app)≈30 μM,Ac-Phe-Arg-AMC (KMaPP 20 EM), Z-Phe-Arg-AMC (K_(M) ^(app)≈40 μM),Z-Leu-Arg-AMC (K_(M) ^(app)≈2 μM); Bz-Phe-Val-Arg-AMC (K_(M) ^(app)≈150μM) respectively. In each case the substrate concentration used in eachassay was equivalent to the K_(M) ^(app). The rate of conversion ofsubstrate to product was derived from the slope of the increase influorescence monitored continuously over time.

[0577] Trypsin Peptidase Activity Assays

[0578] Human pancreatic trypsin (iodination grade; CN Biosciences)activity assays were carried out in 10 mM HEPES, pH 8.0 containing 5 mMCaCl₂ using 0.1 nM trypsin. For the inhibition assays, Z-Gly-Gly-Arg-AMC(K_(M) ^(app)=84 μM) was used as the substrate at a concentrationequivalent to K_(M) ^(app). The rate of conversion of substrate toproduct was derived from the slope of the increase in fluorescencemonitored continuously over time.

[0579] Clostripain Peptidase Activity Assays

[0580] Clostripain (Sigma) activity assays were carried out in 10 mMBTP, pH 6.5 containing 1 mM EDTA, 5 mM 2-mercaptoethanol and 1 mM CaCl₂using 0.3 nM enzyme. For the inhibition assays, Z-Gly-Gly-Arg-AMC (K_(M)^(app)≈100 μM was used as the substrate at a concentration equivalent toK_(M) ^(app). The rate of conversion of substrate to product was derivedfrom the slope of the increase in fluorescence monitored continuouslyover time.

[0581] Calpain Peptidase Activity Assays

[0582] Calpain (human erythrocyte μ-calpain and porcine kidneym-calpain; CN Biosciences) activity assays were carried out in 10 mMHEPES, pH 7.5 containing 2 mM 2-mercaptoethanol and CaCl₂ using 25 nM ofeither enzyme (Sasaki, et. al., J. Biol. Chem., 259, 12489-12494, 1984).For μ-calpain inhibition assays, the buffer contained 100 μM CaCl₂ andAbz-Ala-Asn-Leu-Gly-Arg-Pro-Ala-Leu-Tyr(NO₂Asp-NH₂ (K_(M) ^(app)≈20 μM;Incenta Limited) was used as the substrate. For m-calpain inhibitionassays, the assay buffer contained 200 μM CaCl₂ andAbz-Lys-Leu-Cys(Bzl)-Phe-Ser-Lys-Gln-Tyr(NO₂Asp-NH₂ (K_(M) ^(app)=22 μM;Incenta Limited) was used as the substrate. In both cases the substrateconcentration employed in the assays was equivalent to the K_(M) ^(app).The rate of conversion of substrate to product was derived from theslope of the increase in fluorescence monitored continuously over time.

[0583] Extracellular S. aureus V8 Cysteine Peptidase (staphylopain)Peptidase Activity Assays

[0584]S. aureus V8 was obtained from Prof. S. Arvidson, KarolinskaInstitute, Stockholm, Sweden. Extracellular S. aureus V8 cysteinepeptidase (staphylopain) activity assays were carried out usingpartially purified S. aureus V8 culture supernatant (obtained from Dr.Peter Lambert, Aston University, Birmingham, U.K.). Activity assays werecarried out in 10 mM BTP, pH 6.5 containing 1 mM EDTA, 5 mM2-mercaptoethanol and 1 mM CaCl₂ using two-times diluted partiallypurified extract. For the inhibition assays,Abz-Ile-Ala-Ala-Pro-Tyr(NO₂)-Glu-NH₂ (K_(M) ^(app)≈117 μM; IncentaLimited) was used as the substrate at a concentration equivalent toK_(M) ^(app). The rate of conversion of substrate to product was derivedfrom the slope of the increase in fluorescence monitored continuouslyover time.

[0585] Foot-And-Mouth Disease Leader Peptidase (FMDV-LP) Activity Assays

[0586] Recombinant wild-type FMDV-LP was obtained from Dr. Tim Skern(Institut für Medizinische Biochemie, Abteilung für Biochemie,Universtit Wien, Wien, Austria). Activity assays were carried out in 50mM trisacetate, pH 8.4 containing 1 mM EDTA, 10 mM L-cysteine and 0.25%(w/v) CHAPS using 10 nM enzyme. For the inhibition assays,Abz-Arg-Lys-Leu-Lys-Gly-Ala-Gly-Ser-TyrNO₂)-Glu-NH₂ (K_(M) ^(app)≈51 μM,Incenta Limited) was used as the substrate at a concentration equivalentto K_(M) ^(app). The rate of conversion of substrate to product wasderived from the slope of the increase in fluorescence monitoredcontinuously over time.

[0587] Caspase Peptidase Activity Assays

[0588] Caspases 1-10 were obtained from CN Biosciences or BioVision Inc.(Mountain View, Calif., USA) and all assays were carried out in 50 mMHEPES; pH 7.2, 10% (v/v) glycerol, 0.1% (w/v) CHAPS, 142 mM NaCl, 1 mMEDTA, 5 mM dithiothreitol (DIT) using 0.1-1 U per assay. For caspase 1,Ac-Leu-Glu-His-Asp-AMC was used as the substrate; for caspase 2,Z-Val-Asp-Val-Ala-Asp-AFC was used as the substrate; for caspase 3,Ac-Asp-Glu-Val-Asp-AMC was used as the substrate; for caspase 4,Suc-Tyr-Val-Ala-Asp-AMC was used as the substrate; for caspase 5,Ac-Leu-Glu-His-Asp-AMC was used as the substrate; for caspase 6,Ac-Val-Glu-Ile-Asp-AMC was used as the substrate; for caspase 7,Ac-Asp-Glu-Val-Asp-AMC was used as the substrate; for caspase 8,Ac-Ile-Glu-Thr-Asp-AMC was used as the substrate; for caspase 9,Ac-Leu-Glu-His-Asp-AMC was used as the substrate; for caspase 10,Ac-Ile-Glu-Thr-Asp-AMC was used as the substrate (Nicholson, D. W. andThomberry, N. A., TIBS, 2, 299-306, 1997; Stennicke, H. R. and Salvesen,G. S., J. Biol. Chem., 272(41), 25719-25723, 1997; Talanian, R. V., et.al., J. Biol. Chem., 272(15), 9677-9682, 1997; Wolf, B. B. and Green, D.R., J. Biol. Chem., 274(29), 20049-20052, 1999). The rate of conversionof substrate to product was derived from the slope of the increase influorescence monitored continuously over time.

[0589] Measurement of the Apparent Macroscopic Binding (Michaelis)Constants (K_(M) ^(app)) for Substrates

[0590] The apparent macroscopic binding constant (K_(M) ^(app)) for eachsubstrate was calculated, from the dependence of enzyme activity as afunction of substrate concentration. The observed rates were plotted onthe ordinate against the related substrate concentration on the abscissaand the data fitted by direct regression analysis (Prism v 3.02;GraphPad, San Diego, USA) using Equation 1 (Cornish-Bowden, A.Fundamentals of enzyme kinetics Portland Press; 1995, 93-128.).$\begin{matrix}{v_{i} = \frac{V_{\max}^{app} \cdot \left\lbrack S_{o} \right\rbrack}{\left\lbrack S_{o} \right\rbrack + K_{M}^{app}}} & (1)\end{matrix}$

[0591] In Equation 1 ‘v_(i)’ is the observed initial rate, ‘V_(max)^(app)’ is the observed maximum activity at saturating substrateconcentration, ‘K_(M) ^(app)’ is the apparent macroscopic binding(Michaelis) constant for the substrate, ‘[S_(o)]’ is the initialsubstrate concentration.

[0592] Measurement of the Inhibition Constants

[0593] The apparent inhibition constant (K_(i)) for each compound wasdetermined on the basis that inhibition was reversible and occurred by apure-competitive mechanism. The K_(i) values were calculated, from thedependence of enzyme activity as a function of inhibitor concentration,by direct regression analysis (Prism v 3.02) using Equation 2(Cornish-Bowden, A., 1995.). $\begin{matrix}{v_{i} = \frac{V_{\max}^{app} \cdot \lbrack S\rbrack}{\lbrack S\rbrack + \left\{ {K_{M}^{app} \cdot \left( {\lbrack I\rbrack \text{/}K_{i}} \right)} \right\}}} & (2)\end{matrix}$

[0594] In Equation 2 ‘v_(i)’ is the observed residual activity, ‘V_(max)^(app)’ is the observed maximum activity (i.e. in the absence ofinhibitor), ‘K_(M) ^(app)’ is the apparent macroscopic binding(Michaelis) constant for the substrate, ‘[S]’ is the initial substrateconcentration, ‘K_(i)’ is the apparent dissociation constant and ‘[I]’is the inhibitor concentration.

[0595] In situations where the apparent dissociation constant (K_(i)^(app)) approached the enzyme concentrations, the K_(i) ^(app) valueswere calculated using a quadratic solution in the form described byEquation 3 (Morrison, J. F. Trends Biochem. Sci., 7, 102-105, 1982;Morrison, J. F. Biochim. Biophys. Acta, 185, 269-286, 1969; Stone, S. R.and Hofsteenge, J. Biochemistry, 25, 4622-4628, 1986). $\begin{matrix}{v_{i} = \frac{F\left\{ {E_{o} - I_{o} - K_{i}^{app} + \sqrt{{\left( {E_{o} - I_{o} - K_{i}^{app}} \right)^{2} + {4 \cdot K_{i}^{app} \cdot E_{o}}}}} \right\}}{2}} & (3)\end{matrix}$

 K _(i) ^(app) =K _(i)(1+[S _(o) ]/K _(M) ^(app))  (4)

[0596] In Equation 3 ‘v₁’ is the observed residual activity, ‘F’ is thedifference between the maximum activity (i.e. in the absence ofinhibitor) and minimum enzyme activity, ‘E_(o)’ is the total enzymeconcentration, ‘K_(i) ^(app)’ is the apparent dissociation constant and‘I_(o)’ is the inhibitor concentration. Curves were fitted by non-linearregression analysis (Prism) using a fixed value for the enzymeconcentration. Equation 4 was used to account for the substratekinetics, where ‘K_(i)’ is the inhibition constant, ‘[S_(o)]’ is theinitial substrate concentration and ‘K_(M) ^(app)]’ is the apparentmacroscopic binding (Michaelis) constant for the substrate (Morrison,1982).

[0597] The Second-Order Rate of Reaction of Inhibitor with Enzyme

[0598] Where applicable, the concentration dependence of the observedrate of reaction (k_(obs)) of each compound with enzyme was analysed bydetermining the rate of enzyme inactivation under pseudo-first orderconditions in the presence of substrate (Morrison, J. F., TIBS, 102-105,1982; Tian, W. X. and Tsou, C. L., Biochemistry, 21, 1028-1032, 1982;Morrison, J. F. and Walsh, C. T., from Meister (Ed.), Advances inEnzmol., 61, 201-301, 1988; Tsou, C. L., from Meister (Ed.), Advances inEnzymol., 61, 381-436, 1988;). Assays were carried out by addition ofvarious concentrations of inhibitor to assay buffer containingsubstrate. Assays were initiated by the addition of enzyme to thereaction mixture and the change in fluorescence monitored over time.During the course of the assay less than 10% of the substrate wasconsumed. $\begin{matrix}{F = {{v_{s}t} + \frac{\left( {v_{o} - v_{s}} \right)\left\lfloor {1 - ^{({k_{obs} \cdot t})}} \right\rfloor}{k_{obs}} + D}} & (5)\end{matrix}$

[0599] The activity fluorescence progress curves were fitted bynon-linear regression analysis (Prism) using Eq. 5 (Morrison, 1969;Morrison, 1982); where ‘F’ is the fluorescence response, ‘t’ is time,‘v_(o)’ is the initial velocity, ‘v_(s)’ is the equilibrium steady-statevelocity, ‘k_(obs)’ is the observed pseudo first-order rate constant and‘D’ is the intercept at time zero (i.e. the ordinate displacement of thecurve). The second order rate constant was obtained from the slope ofthe line of a plot of k_(obs) versus the inhibitor concentration (i.e.k_(obs)/[I]). To correct for substrate kinetics, Eq. 6 was used, where‘[S_(o)]’ is the initial substrate concentration and ‘K_(M) ^(app)’ isthe apparent macroscopic binding (Michaelis) constant for the substrate.$\begin{matrix}{k_{inact} = \frac{k_{obs}\left( {1 + {\left\lbrack S_{o} \right\rbrack/K_{M}^{app}}} \right)}{\lbrack I\rbrack}} & (6)\end{matrix}$

[0600] Compounds of the invention were tested by the above describedassays and observed to exhibit cruzipain inhibitory activity orinhibitory activity against an alternative CA C1 cysteine protease withan in vitro Ki inhibitory constant of less than or equal to 100 μM.Exemplary inhibition data for a number of example compounds of theinvention are given in table 2. TABLE 2 Exemplary inhibition data (Kiexpressed as μM). Bovine Human Human EXAMPLE N° Cruzipain Cathepsin SCathepsin L Cathepsin K 22 <5 >50 >100 >5 46 >10 >20 >5 <1 49 >100 >100<10 >100 4 >100 <10 >100 >100

1. A compound according to general formula (I):

wherein: R¹=C₀₋₇-alkyl (when C=0, R¹ is simply hydrogen),C₃₋₆-cycloalkyl or Ar—C₀₋₇-alkyl (when C=0, R¹ is simply an aromaticmoiety Ar); R²=C₁₋₇-alkyl, C₃₋₆-cycloalkyl or Ar—C₀₋₇-alkyl; Y=CHR³—COor CR³R⁴—CO where R³ and R⁴ are independently chosen from C₀₋₇-alkyl,C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl, or Y represents

where L is a number from one to four and R⁵ and R⁶ are independentlychosen from CR⁷R⁸ where R⁷ and R⁸ are independently chosen fromC₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl or halogen; and for each R⁵and R⁶ either R⁷ or R⁸ (but not both R⁷ and R⁸) may additionally bechosen from O—C₀₋₇-alkyl, O—C₃₋₆-cycloalkyl, O—Ar—C₀₋₇-alkyl,S—C₀₋₇-alkyl, S—C₃₋₆-cycloalkyl, S—Ar—C₀₋₇-alkyl, NH—C₀₋₇-aly,NH—C₃₋₆-cycloalkyl, NH—Ar—C₀₋₇-alkyl, N—(C₀₋₇-alkyl)₂,N—(C₃₋₆-cycloalkyl)₂, and N-(Ar—C₀₋₇-alkyl)₂; (X)_(o)=CR⁹R¹⁰, where R⁹and R¹⁰ are independently chosen from C₀₋₇-alkyl, C₃₋₆-cycloalkyl andAr—C₀₋₇-alkyl and o is a number from zero to three; (W)_(n)=O, S, C(O),S(O) or S(O)₂ or, when o is one or greater, NR¹¹, where R¹¹ is chosenfrom C₀₋₇-alkyl, C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl and n is zero or one;(V)_(m)=C(O), C(S), S(O), S(O)₂, S(O)₂NH, OC(O), NHC(O), NHS(O),NHS(O)₂, OC(O)NH, C(O)NH or CR¹²R¹³, where R¹² and R¹³ are independentlychosen from C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl and m is a numberfrom zero to three, provided that when m is greater than one, (V)_(m)contains a maximum of one carbonyl or sulphonyl group; Z=0 (in whichcase compounds of general formula (I) may be named as(2-alkyl-4-oxo-tetrahydrofuran-3-yl)amides), S (in which case compoundsof general formula (I) may be named as(2-alkyl-4-oxo-tetrahydrothiophen-3-yl)amides), or CH₂ (in which casecompounds of general formula (I) may be named as(2-alkyl-5-oxocyclopentyl)amides); U=a stable 5- to 7-memberedmonocyclic or a stable 8- to 11-membered bicyclic ring which is eithersaturated or unsaturated and which includes zero to four heteroatoms (asdetailed below):

wherein R¹⁴ is chosen from: C₀₋₇-alkyl, C₃₋₆-cycloalkyl, Ar—C₀₋₇-alkyl,halogen, O-C₀₋₇-alkyl, O—C₃₋₆-cycloalkyl, O—Ar—C₀₋₇-alkyl, S—C₀₋₇-alkyl,S—C₃₋₆-cycloalkyl, S—Ar—C₀₋₇-alkyl, NH—C₀₋₇-al, NH—C₃₋₆-cycloalkyl,NH—Ar—C₀₋₇-alkyl, N—(C₀₋₇-alkyl)₂, N—(C₃₋₆-cycloalkyl)₂ andN-(Ar—C0-7-alkyl)₂; A is chosen from: CH₂, CHR¹⁴, O, S and NR¹⁵, whereR¹⁴ is as defined above and where R¹⁵ is chosen from C₀₋₇-alkyl,C₃₋₆-cycloalkyl and Ar—C₀₋₇-alkyl; B, D and G are independently chosenfrom: CR¹⁴, where R¹⁴ is as defined above, or N; E is chosen from: CH₂,CHR¹⁴, O, S and NR¹⁵, where R¹⁴ and R¹⁵ are defined as above; J, L, M,R, T, T₂, T₃ and T₄ are independently chosen from: CR¹⁴ and N, where R¹⁴is as defined above; T₅ is chosen from: CH or, only when m+n+o≧1, T₅ mayadditionally be N; q is a number from one to three, thereby defining a5-, 6- or 7-membered ring.
 2. A compound as claimed in claim 1 whereinR¹ comprises C₀₋₇-alkyl or Ar—C₀₋₇-alkyl.
 3. A compound as claimed inclaim 2 wherein R¹ is selected from hydrogen or one of the followingmoieties:


4. A compound as claimed in any one of claims 1 to 3 wherein R² isC₁₋₇-alkyl or Ar—C₀₋₇-alkyl.
 5. A compound as claimed in claim 4 whereinR² is selected from one of the following moieties:

wherein R¹⁴ and R¹⁵ are as defined previously.
 6. A compound as claimedin any one of claims 1 to 5 in which Z represents an oxygen atom.
 7. Acompound as claimed in any one of claims 1 to 6 wherein Y is 0HR⁴COwhere R⁴ is selected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl; or wherein Ycomprises a group:

where R⁵ and R⁶ are each CR⁷R⁸ and each R⁷ and R⁸ is, independently,selected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl.
 8. A compound as claimed inany one of claims 1 to 6, wherein Y is CHR⁴CO where R⁴ is selected fromC₃₋₆-cycloalkyl.
 9. A compound as claimed in claim 1 wherein Y isselected from one of the following moieties:

wherein R¹⁴ and R¹⁵ and Ar are as defined previously.
 10. A compound asclaimed in any one of claims 1 to 6, wherein Y is CHR⁴CO where R⁴ isAr—CH₂—, where the aromatic ring is an optionally substituted phenyl ormonocyclic heterocycle.
 11. A compound as claimed in any one of claims 1to 6, wherein Y is CHR⁴CO where R⁴ represents a simple branched alkylgroup or a straight heteroalkyl chain.
 12. A compound as claimed in anyone of claims 1 to 6, wherein Y is CHR⁴CO where the R⁴ group comprisescyclohexylmethyl.
 13. A compound as claimed in any one of claims 1 to 6,wherein Y is selected from the following:

wherein R¹⁴ and Ar are as defined previously.
 14. A compound as claimedin any one of claims 1 to 13 wherein, in the group (X)_(o), X is CR⁸R⁹and each of R⁹ and R¹⁰ is selected from C₀₋₇-alkyl or Ar—C₀₋₇-alkyl 15.A compound as claimed in any one of claims 1 to 14, wherein (X)_(o) isone of the following moieties:

wherein R¹⁴ and R¹⁵ are as defined previously.
 16. A compound as claimedin any one of claims 1 to 14, wherein (X)_(o) a simple alkyl group andwhere o=0 or
 1. 17. A compound as claimed in any one of claims 1 to 16wherein, in the group (W)_(n): W is O, S, SO₂, SO, C(O) or when o is oneor greater, NR¹¹, where R¹¹ is C₀₋₄-alkyl; and n is 0 or
 1. 18. Acompound as claimed in any one of claims 1 to 16 wherein, in the group(W)_(n): W is O, S, SO₂, C(O) and n is 0 or
 1. 19. A compound as claimedin any one of claims 1 to 18 wherein, in the group (V)_(m): V is C(O),C(O)NH or CHR¹³, where R¹³ is C₀₋₄-alkyl; and m is 0 or
 1. 20. Acompound as claimed in any one of claims 1 to 19 wherein the combination(V)_(m) and (W)_(m) is one of the following:


21. A compound as claimed in any one of claims 1 to 19 wherein thecombination (X)₀, (V)_(m) and (W)_(m) is one of the following:


22. A compound as claimed in any one of claims 1 to 21 wherein Ucomprises an optionally substituted 5- or 6-membered saturated orunsaturated heterocycle or an optionally substituted saturated orunsaturated 9- or 10-membered heterocycle.
 23. A compound as claimed inclaim 22 wherein U comprises one of the following:

wherein R¹⁴ is as defined previously.
 24. A compound as claimed in anyone of claims 1 to 21 wherein U comprises a bulky alkyl or aryl group atthe para position of an aryl Ar.
 25. A compound as claimed in any one ofclaims 1 to 21 wherein U comprises a meta or para-biaryl Ar—Ar, where Aris as previously defined.
 26. A compound as claimed in any one of claims1 to 21 wherein U comprises a 6,6 or 6,5 or 5,6-fused aromatic ring. 27.A compound as claimed in any one of claims 1 to 21, wherein U representsa group:

wherein R¹⁴, D, E, G, J, L, M, R, T, T₂, T₃ and T₄ are as definedpreviously.
 28. A compound as claimed in any one of claims 1 to 21,wherein U represents a group

wherein R¹⁴, D, E, G, J, L, M, R and T are as defined previously.
 29. Acompound as claimed in any one of claims 1 to 21, wherein U represents agroup

wherein R¹⁴, D, E, G, J and L are as defined previously.
 30. A method ofvalidating a known or putative cysteine protease inhibitor as atherapeutic target, the method comprising: (a) assessing the in vitrobinding of a compound as claimed in any one of claims 1 to 29 to anisolated known or putative cysteine protease, providing a measure of‘potency’; and optionally, one or more of the steps of: (b) assessingthe binding of a compound as claimed in any one of claims 1 to 29 toclosely related homologous proteases of the target and generalhouse-keeping proteases (e.g. trypsin) to provides a measure of‘selectivity’; (c) monitoring a cell-based functional marker of aparticular cysteine protease activity, in the presence of a compound asclaimed in any one of claims 1 to 29; and (d) monitoring an animalmodel-based functional marker of a particular cysteine proteaseactivity, in the presence of a compound as claimed in any one of claims1 to
 29. 31. The use of a compound as claimed in any one of claims 1 to29 in the validation of a known or putative cysteine protease inhibitoras a therapeutic target.
 32. A compound as claimed in any one of claims1 to 29 for use in medicine, especially for preventing or treatingdiseases in which the disease pathology may be modified by inhibiting acysteine protease.
 33. The use of a compound as claimed in any one ofclaims 1 to 29 in the preparation of a medicament for preventing ortreating diseases in which the disease pathology may be modified byinhibiting a cysteine protease.
 34. A compound as claimed in any one ofclaims 1 to 29 for use in preventing or treating Chagas' disease.
 35. Acompound as claimed in any one of claims 24 to 29 for use in preventingor treating Chagas' disease.
 36. The use of a compound as claimed in anyone of claims 1 to 29 in the preparation of a medicament for preventingor treating Chagas' disease.
 37. The use of a compound as claimed in anyone of claims 24 to 29 in the preparation of a medicament for preventingor treating Chagas' disease.
 38. A pharmaceutical or veterinarycomposition comprising one or more compounds as claimed in any one ofclaims 1 to 29 and a pharmaceutically or veterinarily acceptablecarrier.
 39. A process for the preparation of a pharmaceutical orveterinary composition as claimed in claim 38, the process comprisingbringing the active compound(s) into association with the carrier, forexample by admixture.